Use of plant fatty acyl hydroxylases to produce hydroxylated fatty acids and derivatives in plants

ABSTRACT

The present invention relates to the identification of nucleic acid sequences and constructs, and methods related thereto, and the use of these sequences and constructs to produce genetically modified plants for the purpose of altering the composition of plant oils, waxes and related compounds.

This Invention was made with Government support under Contract No.DE-FG02-94ER20133 awarded by the Department of Energy. The Governmenthas certain rights in this invention.

TECHNICAL FIELD

The present invention concerns the identification of nucleic acidsequences and constructs, and methods related thereto, and the use ofthese sequences and constructs to produce genetically modified plantsfor the purpose of altering the composition of plant oils, waxes andrelated compounds.

BACKGROUND

Extensive surveys of the fatty acid composition of seed oils fromdifferent species of higher plants have resulted in the identificationof more than 210 naturally occurring fatty acids which differ by thenumber and arrangement of double or triple bonds and various functionalgroups, such as hydroxyls, ketones, epoxys, cyclopentenyl or cyclopropylgroups, furans or halogens (van de Loo et al. 1993). At least 33structurally distinct monohydroxylated plant fatty acids, and 12different polyhydroxylated fatty acids have been described (reviewed byvan de Loo et al. 1993; Smith, 1985).

The most commonly occurring fatty acids in both membrane and storagelipids are 16- and 18-carbon fatty acids which may have from zero tothree, methylene-interrupted, unsaturations. These are synthesized fromthe fully saturated species as the result of a series of sequentialdesaturations which usually begin at the Δ9 carbon and progress in thedirection of the methyl carbon (Browse and Somerville, 1991). Fattyacids which cannot be described by this simple algorithm are generallyconsidered "unusual" even though several, such as lauric (12:0), erucic(22:1) and ricinoleic acid (12D-hydroxyoctadec-cis-9-enoic acid) are ofsignificant commercial importance. The biosynthesis of hydroxylatedfatty acids such as ricinoleic acid in castor (Ricinus communis) seed isthe subject of this invention.

The taxonomic relationships between plants having similar or identicalkinds of unusual fatty acids have been examined (van de Loo et al.,1993). In some cases, particular fatty acids occur mostly or solely inrelated taxa. In other cases there does not appear to be a direct linkbetween taxonomic relationships and the occurrence of unusual fattyacids. In this respect, ricinoleic acid has now been identified in 12genera from 10 families (reviewed in van de Loo et al., 1993). Thus, itappears that the ability to synthesize hydroxylated fatty acids hasevolved several times independently during the radiation of theangiosperms. This suggested to us that the enzymes which introducehydroxyl groups into fatty acids arose by minor modifications of arelated enzyme. Indeed, as noted below, this invention is based on ourdiscovery that plant fatty acid hydroxylases are highly homologous toplant fatty acid desaturases.

A feature of hydroxylated or other unusual fatty acids is that they aregenerally confined to seed triacylglycerols, being largely excluded fromthe polar lipids by unknown mechanisms (Battey and Ohlrogge 1989; Prasadet al., 1987). This is particularly intriguing since diacylglycerol is aprecursor of both triacylglycerol and polar lipid. With castormicrosomes, there is some evidence that the pool ofricinoleoyl-containing polar lipid is minimized by a preference ofdiacylglycerol acyltransferase for ricinoleate-containingdiacylglycerols (Bafor et al. 1991). Analyses of vegetative tissues havegenerated few reports of unusual fatty acids, other than those occurringin the cuticle. A small number of exceptions exist in which unusualfatty acids are found in tissues other than the seed.

Castor (Ricinus communis L.) is a minor oilseed crop. Approximately 50%of the seed weight is oil (triacylglycerol) in which 85-90% of totalfatty acids are the hydroxylated fatty acid, ricinoleic acid(12D-hydroxyoctadec-cis-9-enoic acid). Oil pressed or extracted fromcastor seeds has many industrial uses based upon the properties endowedby the hydroxylated fatty acid. The most important uses are productionof paints and varnishes, nylon-type synthetic polymers, resins,lubricants, and cosmetics (Atsmon 1989). In addition to oil, the castorseed contains the extremely toxic protein ricin, allergenic proteins,and the alkaloid ricinine. These constituents preclude the use of theuntreated seed meal (following oil extraction) as a livestock feed,normally an important economic aspect of oilseed utilization.Furthermore, with the variable nature of castor plants and a lack ofinvestment in breeding, castor has few favorable agronomiccharacteristics. For a combination of these reasons, castor is no longergrown in the United States and the development of an alternativedomestic source of hydroxylated fatty acids would be attractive. Theproduction of ricinoleic acid, the important constituent of castor oil,in an established oilseed crop through genetic engineering would be aparticularly effective means of creating a domestic source.

The biosynthesis of ricinoleic (12D-hydroxyoctadec-cis-9-enoic) acidfrom oleic acid in the developing endosperm of castor (Ricinus communis)has been studied by a variety of methods. Morris (1967) established indouble-labeling studies that hydroxylation occurs directly by hydroxylsubstitution rather than via an unsaturated-, keto- orepoxy-intermediate. Hydroxylation using oleoyl-CoA as precursor can bedemonstrated in crude preparations or microsomes, but activity inmicrosomes is unstable and variable, and isolation of the microsomesinvolved a considerable, or sometimes complete loss of activity(Galliard and Stumpf, 1966; Moreau and Stumpf, 1981. Oleic acid canreplace oleoyl-CoA as a precursor, but only in the presence of CoA, Mg²⁺and ATP (Galliard and Stumpf, 1966) indicating that activation to theacyl-CoA is necessary. However, no radioactivity could be detected inricinoleoyl-CoA (Moreau and Stumpf, 1981). These and more recentobservations (Bafor et al., 1991) have been interpreted as evidence thatthe substrate for the castor oleate hydroxylase is oleic acid esterifiedto phosphatidylcholine or another phospholipid.

The hydroxylase is sensitive to cyanide and azide, and dialysis againstmetal chelators reduces activity, which could be restored by addition ofFeSO₄, suggesting iron involvement in enzyme activity (Galliard andStumpf, 1966). Ricinoleic acid synthesis requires molecular oxygen(Galliard and Stumpf, 1966; Moreau and Stumpf 1981) and requires NAD(P)Hto reduce cytochrome b5 which is thought to be the intermediate electrondonor for the hydroxylase reaction (Smith et al., 1992). Carbon monoxidedoes not inhibit hydroxylation, indicating that a cytochrome P450 is notinvolved (Galliard and Stumpf, 1966; Moreau and Stumpf 1981). Data froma study of the substrate specificity of the hydroxylase show that allsubstrate parameters (i.e. chain length and double bond position withrespect to both ends) are important; deviations in these parameterscaused reduced activity relative to oleic acid (Howling et al., 1972).The position at which the hydroxyl was introduced, however, wasdetermined by the position of the double bond, always being threecarbons distal. Thus, the castor acyl hydroxylase enzyme can produce afamily of different hydroxylated fatty acids depending on theavailability of substrates. Thus, although we refer to the enzymethroughout as oleate hydroxylase it can more properly be considered anacyl hydroxylase of broad substrate specificity.

The only other organism in which ricinoleic acid biosynthesis has beeninvestigated is the ergot fungus, Claviceps purpurea. Ricinoleateaccumulates (up to 40% of the fatty acids) in the glycerides producedparticularly by sclerotia of anaerobic cultures (Kren et al., 1985). Asthis suggests, oxygen is not necessary for the synthesis of ricinoleicacid in Claviceps, and the precursor of ricinoleic acid in fact appearsto be linoleic acid (Morris et al., 1966). However, ricinoleic acid maynot be formed simply by hydration of linoleic acid, since there are nofree hydroxyl groups in ergot oil. Rather, the hydroxyl groups are allesterified to other, non-hydroxy fatty acids, leading to a range oftetra-acyl-, penta-acyl- and hexa-acyl-glycerides. These estolides maybe formed by a direct enzymic addition of non-hydroxy fatty acids acrossthe A12 double bond of linoleate (Morris, 1970). Ricinoleic acid may,therefore, be merely an artifact of the hydrolysis employed to study thefatty acid composition of the oil.

The castor oleate hydroxylase has many superficial similarities to themicrosomal fatty acyl desaturases (Browse and Somerville, 1991). Inparticular, plants have a microsomal oleate desaturase active at the Δ12position. The substrate of this enzyme (Schmidt et al., 1993) and of thehydroxylase (Bafor et al., 1991) appears to be oleate esterified to thesn-2 position of phosphatidylcholine. The modification occurs at thesame position (Δ12) in the carbon chain, and requires the samecofactors, namely electrons from NADH via cytochrome b₅ (Kearns et al.,1991; Smith et al., 1992) and molecular oxygen. Neither enzyme isinhibited by carbon monoxide (Moreau and Stumpf, 1981) thecharacteristic inhibitor of cytochrome P450 enzymes.

Conceptual Basis of the Invention

A feature of certain fatty acid modifying enzymes such as fatty acyldesaturases and castor oleate hydroxylase is that they catalyzereactions in which an unactivated C--H bond is cleaved. To catalyze thisenergetically demanding cleavage, these fatty acid modifying enzymesutilize the high oxidizing power of molecular oxygen. There arepresently two known classes of enzyme cofactors capable of this type ofO₂ -dependent chemistry. The haem-containing oxygenase includingcytochromes P450 are one class. However, as noted above, substantialevidence indicates that oleate hydroxylase is not a cytochrome P450enzyme. The second class of cofactor known to be capable of this type ofO₂ -dependent chemistry is less well characterized, but is typified bythe bacterial enzyme methane monooxygenase (van de Loo et al., 1993).The cofactor in the hydroxylase component of methane monooxygenase istermed a μ-oxo bridged diiron cluster (FeOFe). The two iron atoms of theFeOFe cluster are liganded by protein-derived nitrogen or oxygen atoms,and are tightly redox-coupled by the covalently-bridging oxygen atom.The catalytic cycle of methane monooxygenase is not so well understoodas that of the P450 oxygenases, but there are known differences andsimilarities. Rather than two discrete single-electron reductions of thehaem cofactor, the FeOFe cluster accepts two electrons, reducing it tothe diferrous state, before oxygen binding. Upon oxygen binding, it islikely that heterolytic cleavage also occurs, leading to a high valentoxoiron reactive species that is very similar to that of the haemcofactor, but stabilized by resonance rearrangements possible within thetightly coupled FeOFe cluster, rather than through a porphyrin-orprotein-derived ligand. The stabilized high-valent oxoiron state ofmethane monooxygenase is capable of proton extraction from methane,followed by oxygen transfer, giving methanol.

The FeOFe cofactor has been shown to be directly relevant to plant fattyacid modifications by the demonstration that castor stearoyl-ACPdesaturase contains this type of cofactor (Fox et al., 1993). Thisdesaturase is a member of a small family of plant fatty acid desaturasesthat are soluble enzymes, whereas most other desaturases aremembrane-bound. Putative iron-binding motifs have been identified in thecastor stearoyl-ACP desaturase primary structure by comparison to othersoluble enzymes containing the FeOFe cluster (Fox et al., 1993). Thesesimilar motifs, (D/E)--E--X--R--H, are characteristically spacedapproximately 90 residues apart in a number of soluble diiron-oxoproteins, including methane monooxygenase. Recently, cDNA clones forseveral plant membrane-bound desaturases encoding microsomal and plastidω-3 and ω-6 desaturases of several plant species have been isolated₋₋(Arondel et al., 1992; Iba et al., 1993; Okuley et al., 1994; Yadav etal., 1993). Of great interest is the identification of a similarlyrepeated motif in all of these sequences (Schmidt et al., 1993), themembrane-bound rat stearoyl-CoA desaturase (Thiede et al., 1986) and intwo membrane-bound monooxygenases (Kok et al., 1989; Suzuki et al.,1991). This motif, H--X--X--H--H in the desaturases and H--X--X--X--H--Hin the monooxygenases, may be the functional equivalent inmembrane-bound FeOFe proteins of the (D/E)--E--X--R--H motif in thesoluble FeOFe proteins. This suggests that the plant membrane bounddesaturases may also accomplish oxygen-dependent fatty acid desaturationthrough an FeOFe cofactor.

Of the well-characterized FeOFe-containing enzymes, methanemonooxygenase catalyses a reaction involving oxygen-atom transfer (CH₄→CH₃ OH), while the FeOFe cluster of ribonucleotide reductase catalysesthe oxidation of tyrosine to form a tyrosyl cation radical withoutoxygen-atom transfer. However, site-directed mutagenesis of Phe208 toTyr resulted in the conversion of this enzyme to an oxygen transfercatalyst, Tyr208 being hydroxylated and shown to be acting as a ligandto one iron of the FeOFe cluster. Therefore, the argument made for theP450 oxygenases catalyzing a range of reactions through the use of thesame reactive intermediate modulated by the electronic and structuralenvironment provided by the protein, can also be applied toFeOFe-containing enzymes. Modifications of the active site of plantfatty acid oxidizing enzymes containing FeOFe clusters could thus alterthe outcome of the reaction, including whether oxygen-atom transferoccurs or not.

On the basis of the foregoing considerations, we hypothesized that thecastor oleate hydroxylase is a structurally modified fatty acyldesaturase, based upon three arguments. The first argument involves thetaxonomic distribution of plants containing ricinoleic acid. Ricinoleicacid has been found in 12 genera of 10 families of higher plants(reviewed in van de Loo et al., 1993). Thus, plants in which ricinoleicacid occurs are found throughout the plant kingdom, yet close relativesof these plants do not contain the unusual fatty acid. This patternsuggests that the ability to synthesize ricinoleic acid has arisenseveral times independently, and is therefore a quite recent divergence.In other words, the ability to synthesize ricinoleic acid has evolvedrapidly, suggesting that a relatively minor genetic change was necessaryto accomplish it. Several mechanisms for such facile evolution of a newenzyme activity are envisaged. One mechanism would be for themodification of a gene normally encoding a fatty acid hydroxylase activein the epidermis and involved in the synthesis of a hydroxy-fatty acidcutin monomer. The other mechanism would be for modification of a geneencoding a microsomal fatty acid desaturase, such that instead ofperforming one type of oxidation reaction (desaturation) it now performsanother (hydroxylation).

The second argument is that many biochemical properties of castoroleate-12-hydroxylase are similar to those of the microsomaldesaturases, as discussed above (eg., both preferentially act on fattyacids esterified to the sn-2 position of phosphatidylcholine, both usecytochrome b5 as an intermediate electron donor, both are inhibited bycyanide, both require molecular oxygen as a substrate, both are thoughtto be located in the endoplasmic reticulum).

The third argument stems from the discussion of oxygenase cofactorsabove, in which it is suggested that the plant membrane bound fatty aciddesaturases may have a μ-oxo bridged diiron cluster-type cofactor, andthat such cofactors are capable of catalyzing both fatty aciddesaturations and hydroxylations, depending upon the electronic andstructural properties of the protein active site.

Taking these three arguments together, it was hypothesized thatoleate-12-hydroxylase of castor endosperm is homologous to themicrosomal oleate Δ12 desaturase found in all plants. When thisinvention was conceived, the structure of microsomal oleate Δ12desaturase (also known as ω-6 desaturase) was not known. However, basedon the high degree of homology between plastid andendoplasmic-reticulum-localized ω-3 desaturases (Iba et al., 1993), wefurther hypothesized that the microsomal Δ12 desaturase was homologousto the microsomal (ω-3) desaturase in particular, and also to theequivalent desaturases of the chloroplast inner envelope. A number ofgenes encoding microsomal ω-3 desaturases from various species haverecently been cloned and substantial information about the structure ofthese enzymes is now known (Arondel et al., 1992; Iba et al., 1993; vande Loo and Somerville, 1993; Yadav et al., 1993). Hence in the followinginvention we teach how to use structural information about fatty acyldesaturases to isolate fatty acyl hydroxylase genes. Although, in thefollowing example we reduce this invention to practice only for thecastor oleate hydroxylase, this example unequivocally teaches the methodby which any carbon-monoxide insensitive plant fatty acyl hydroxylasegene can be identified by one skilled in the art.

The invention is more fully described by reference to the following:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Autoradiogram of a filter containing castor cDNA clones from96-well plates #28-36 which had been replicated in a 3×3 grid and probedwith ³² P-labelled cDNA from developing castor seeds. The positions ofthe wells in the original 96-well plates is indicated by the numbers andletters along the edges. The position of clones from each 96-well platerelative to other 96-well plates is indicated in the box at the lowerright corner.

FIG. 2. Autoradiogram of a filter containing castor cDNA clones from96-well plates #28-36 which had been replicated in a 3×3 grid and probedwith ³² P-labelled cDNA from developing castor leaves. The positions ofthe wells in the original 96-well plates is indicated by the numbers andletters along the edges. The position of clones from each 96-well platerelative to other 96-well plates is indicated in the box at the lowerright corner.

FIG. 3. Autoradiogram of a filter containing castor cDNA clones from96-well plates #28-36 which had been replicated in a 3×3 grid and probedwith ³² P-labelled DNA from redundant clones sequenced in batch 1. Thepositions of the wells in the original 96-well plates is indicated bythe numbers and letters along the edges. The position of clones fromeach 96-well plate relative to other 96-well plates is indicated in thebox at the lower right corner.

FIG. 4. Partial uncorrected nucleotide sequences of castor cDNA clonepCRS677 (SEQ ID NO:1).

FIG. 5. Partial uncorrected nucleotide sequences of castor cDNA clonepCRS834 (SEQ ID NO:2).

FIGS. 6a and 6b. Abbreviated results from BLASTX-mediated comparison(SEQ ID NOS: 4, 6, 8, 10, 12 and 14) of all six translations (SEQ IDNOs: 3, 5, 7, 9, 11 and 13) of the partial nucleotide sequence ofpCRS677 (FIG. 4) with the public sequence databases. The result showsthat pCRS677 exhibits significant deduced amino acid sequence homologyto an ω-3 desaturase from Brassica napus.

FIGS. 7a and 7b. Abbreviated results from BLASTX-mediated comparison(SEQ ID NOs: 16, 18, 20, 22, 24 and 26) of all six translations (SEQ IDNos: 15, 17, 19, 21, 23 and 25) of the partial nucleotide sequence ofpCRS834 (FIG. 5) with the public sequence databases. The result showsthat pCRS834 exhibits significant deduced amino acid sequence homologyto an ω-3 desaturase from Brassica napus.

FIGS. 8a and 8b. Comparison of the partial (uncorrected) nucleotidesequences of pCRS677 and pCRS834 (SEQ ID NOs: 27 and 28, respectively).

FIG. 9. Comparison of partial nucleotide sequences of ten castor cDNAclones (SEQ ID Nos: 29-38, respectively).

FIG. 10a and 10b. Nucleotide sequence of cDNA insert in pFL2 (SEQ. ID.NO. 39) and the deduced amino acid sequence (SEQ. ID. NO. 40) in singleletter code. The positions of the putative iron-binding sites arehighlighted.

FIG. 11. Comparison of deduced amino acid sequences of the cDNA insertin pFL2 and the Arabidopsis fad2 cDNA clone encoding (SEQ ID Nos: 41 and42) an ω-6 fatty acyl desaturase.

FIGS. 12a, 12b and 12c. Northern blot analysis of pFL2 expression incastor. A ³² P-labelled probe corresponding to ˜700 bp of the 3' end ofclone pFL2 was hybridized to poly(A)⁺ RNA from leaves (L) and developingseeds (S) of castor. Panel A: the blot was exposed to film for 30 min.The migration of RNA standards (kb) is shown to the right. Panel B: thesame blot was exposed for 16 h. Panel C: the same blot was hybridized toa ³² P-labelled probe made from the Colletotrichum graminicola β-tubulingene TUB2.

FIG. 13. A Southern blot of genomic DNA from Arabidopsis thaliana andcastor (Ricinus communis) digested with restriction enzymes EcoRI (E),BamHI (B), or HindIII (H), was hybridized at high stringency (65° C.)with the ³² P-labelled insert of clone pFL2. Migration of DNA standards(kb) is shown to the left.

FIG. 14. A Southern blot of genomic DNA from Arabidopsis thaliana andcastor (Ricinus communis) digested with restriction enzymes EcoRI (E),BamHI (B), or HindIII (H), was hybridized at moderate stringency (52°C.) with the ³² P-labelled insert of clone pFL2. Migration of DNAstandards (kb) is shown to the left.

FIGS. 15a, 15b and 15c. Comparison of the nucleotide sequences of thecastor fah12 cDNA insert in pFL2 and the Arabidopsis fad2 cDNA (SEQ IDNos: 43 and 44).

FIG. 16. Map of binary Ti plasmid pBI121.

FIG. 17. Map of binary Ti plasmid pSLJ4K1.

FIG. 18. Mass spectrum of TMS-methyl-ricinoleate

FIGS. 19a and 19b. Fragmentation pattern of TMS-methyl ricinoleate bymass spectrometry.

FIGS. 20a, 20b and 20c. Gas chromatograms of control and transgenictobacco plants. The arrow indicates the peak of methyl-ricinoleate.

FIG. 21. Gas chromatogram of methyl-ricinoleate standard.

FIG. 22. Total ion chromatogram of fatty acids from seeds of 2--2transgenic tobacco plants expressing the fah12 gene. Themethyl-ricinoleate peak is indicated with an arrow.

FIG. 23. Mass spectrum of methyl-ricinoleate peak from peak eluting at14.65 min in FIG. 22.

FIG. 24. Mass spectrum of TMS-methyl ricinoleate standard.

SUMMARY OF THE INVENTION

This invention relates to plant fatty acyl hydroxylases. Methods to useconserved amino acid or nucleotide sequences to obtain plant fatty acylhydroxylases are described. Also described is the use of cDNA clonesencoding a plant hydroxylase to produce hydroxylated fatty acids intransgenic plants.

In a first embodiment, this invention is directed to nucleic acidsequences which encode a plant oleate hydroxylase. This includessequences which encode biologically active plant oleate hydroxylase aswell as sequences which are to be used as probes, vectors fortransformation or cloning intermediates. All or a portion of the aminoacid sequence, the genomic sequence or cDNA sequence of plant oleatehydroxylase is intended.

Of special interest are recombinant DNA constructs which can provide forthe transcription or transcription and translation (expression) of theplant oleate hydroxylase sequence. In particular, constructs which arecapable of transcription or transcription and translation in plant hostcells are preferred. Such constructs may contain a variety of regulatoryregions including transcriptional initiation regions obtained from genespreferentially expressed in plant seed tissue.

In a second aspect, this invention relates to the presence of suchconstructs in host cells, especially plant host cells which have anexpressed plant oleate hydroxylase therein.

In yet a different aspect, this invention relates to a method forproducing a plant oleate hydroxylase in a host cell or progeny thereofvia the expression of a construct in the cell. Cells containing a plantoleate hydroxylase as a result of the production of the plant oleatehydroxylase encoding sequence are also contemplated herein.

In a different embodiment, this invention relates to methods of using aDNA sequence encoding a plant oleate hydroxylase for the modification ofthe proportion of hydroxylated fatty acids produced within a cell,especially plant cells. Plant cells having such a modified hydroxylatedfatty acid composition are also contemplated herein.

In a further aspect of this invention, plant oleate hydroxylase proteinsand sequences which are related thereto, including amino acid andnucleic acid sequences, are contemplated.

Plant oleate hydroxylase exemplified herein includes a Ricinus communis(castor) oleate hydroxylase. This exemplified oleate hydroxylase may beused to obtain other plant fatty acid hydroxylases of this invention.

DETAILED DESCRIPTION OF THE INVENTION

A genetically transformed plant of the present invention whichaccumulates hydroxylated fatty acids can be obtained by expressing thedouble-stranded DNA molecules described in this application.

A plant oleate hydroxylase of this invention includes any sequence ofamino acids, such as a protein, polypeptide or peptide fragment, ornucleic acid sequences encoding such polypeptides, obtainable from aplant source which demonstrates the ability to catalyze the productionof hydroxyoleic acid from CoA, ACP or lipid-linked substrates underplant enzyme reactive conditions. By "enzyme reactive conditions" ismeant that any necessary conditions are available in an environment(i.e., such factors as temperature, pH, lack of inhibiting substances)which will permit the enzyme to function.

Preferential activity of a plant oleate hydroxylase toward a particularfatty acyl substrate is determined upon comparison of hydroxylated fattyacid product amounts obtained per different fatty acyl substrates. Forexample, by "oleate preferring" is meant that the hydroxylase activityof the enzyme preparation demonstrates a preference foroleate-containing substrates over other substrates. Although the precisesubstrate of the oleate desaturase is not known, it is thought to be anoleic acid moiety which is esterified to a phospholipid such asphosphatidylcholine, phosphatidylethanolamine, phosphatidic acid or aneutral lipid such as diacylglycerol or a Coenzyme-A thioester. As notedabove, significant activity has been observed in radioactive labellingstudies using other fatty acyl substrates (Howling et al., 1972)indicating that the substrate specificity is for a family of relatedfatty acyl compounds. Of particular interest, we envision that thecastor oleate hydroxylase may be used for production of12-hydroxy-9-octadecenoic acid (ricinoleate), 12-hydroxy-9-hexadecenoicacid, 14-hydroxy-11-eicosenoic acid, 16-hydroxy-13-docosenoic acid,9-hydroxy-6-octadecenoic acid by expression in plants species whichproduce the non hydroxylated precursors. We also envision production ofadditionally modified fatty acids such as12-hydroxy-9,15-octadecadienoic acid that result from desaturation ofhydroxylated fatty acids (eg., 12-hydroxy-9-octadecenoic acid in thisexample).

As noted above, a plant oleate hydroxylase of this invention willdisplay activity toward fatty acyl substrates. During biosynthesis oflipids in a plant cell, fatty acids are typically covalently bound toacyl carrier protein (ACP), coenzyme A (CoA) or various cellular lipids.Plant oleate hydroxylases which display preferential activity towardlipid-linked acyl substrate are especially preferred because they arelikely to be closely associated with normal pathway of storage lipidsynthesis in immature embryos. However, activity toward acyl-CoAsubstrates or other synthetic substrates, for example, is alsocontemplated herein.

Other plant oleate hydroxylases are obtainable from the specificexemplified sequences provided herein. Furthermore, it will be apparentthat one can obtain natural and synthetic plant oleate hydroxylasesincluding modified amino acid sequences and starting materials forsynthetic-protein modeling from the exemplified plant oleate hydroxylaseand from plant oleate hydroxylases which are obtained through the use ofsuch exemplified sequences. Modified amino acid sequences includesequences which have been mutated, truncated, increased and the like,whether such sequences were partially or wholly synthesized. Sequenceswhich are actually purified from plant preparations or are identical orencode identical proteins thereto, regardless of the method used toobtain the protein or sequence, are equally considered naturallyderived.

Thus, one skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared and used to screen and recover "homologous" or "related" oleatehydroxylases from a variety of plant sources. Typically, nucleic acidprobes are labeled to allow detection, preferably with radioactivityalthough enzymes or other methods may also be used. For immunologicalscreening methods, antibody preparations either monoclonal or polyclonalare utilized. Polyclonal antibodies, although less specific, typicallyare more useful in gene isolation. For detection, the antibody islabeled using radioactivity or any one of a variety of secondantibody/enzyme conjugate systems that are commercially available.

Homologous sequences are found when there is an identity of sequence andmay be determined upon comparison of sequence information, nucleic acidor amino acid, or through hybridization reactions between a known oleatehydroxylase and a candidate source. Conservative changes, such asGlu/Asp, Val/Ile, Ser/Thr, Arg/Lys and Gln/Asn may also be considered indetermining sequence homology. Typically, a lengthy nucleic acidsequence may show as little as 50-60% sequence identity, and morepreferably at least about 70% sequence identity, between the targetsequence and the given plant oleate hydroxylase of interest excludingany deletions which may be present, and still be considered related.Amino acid sequences are considered homologous by as little as 25%sequence identity between the two complete mature proteins. (Seegenerally, Doolittle, R.F., OF URFS and ORFS, University Science Books,Calif., 1986.)

A genomic or other appropriate library prepared from the candidate plantsource of interest may be probed with conserved sequences from the plantoleate hydroxylase to identify homologously related sequences. Use of anentire cDNA or other sequence may be employed if shorter probe sequencesare not identified. Positive clones are then analyzed by restrictionenzyme digestion and/or sequencing. When a genomic library is used, oneor more sequences may be identified providing both the coding region, aswell as the transcriptional regulatory elements of the oleatehydroxylase gene from such plant source. Probes can also be considerablyshorter than the entire sequence. Oligonucleotides may be used, forexample, but should be at least about 10, preferably at least about 15,more preferably at least 20 nucleotides in length. When shorter lengthregions are used for comparison, a higher degree of sequence identity isrequired than for longer sequences. Shorter probes are oftenparticularly useful for polymerase chain reactions (PCR), especiallywhen highly conserved sequences can be identified (See Gould, et al.,1989 for examples of the use of PCR to isolate homologous genes fromtaxonomically diverse species).

When longer nucleic acid fragments are employed (>100 bp) as probes,especially when using complete or large cDNA sequences, one would screenwith low stringencies (for example 40°-50° C. below the meltingtemperature of the probe) in order to obtain signal from the targetsample with 20-50% deviation, i.e., homologous sequences. (Beltz, et al.1983).

In a preferred embodiment, a plant oleate hydroxylase of this inventionwill have at least 67% overall amino acid sequence similarity with theexemplified plant oleate hydroxylase. This level of similarity issufficient to distinguish the castor oleate hydroxylase from theArabidopsis fad2 gene product which encodes a Δ12 (or ω6) desaturase. Inparticular, oleate hydroxylases which are obtainable from an amino acidor nucleic acid sequence of a castor oleate hydroxylase (See, FIG. 10)are especially preferred. The plant oleate hydroxylases may havepreferential activity toward longer or shorter chain fatty acylsubstrates. Plant fatty acyl hydroxylases having oleate-12-hydroxylaseactivity and eicosenoate-14-hydroxylase activity are both consideredhomologously related proteins because of in vitro evidence, noted in theintroduction, that the castor oleate hydroxylase will act on substratesother than oleate. As noted above, hydroxylated fatty acids may besubject to further enzymatic modification by other enzymes which arenormally present or are introduced by genetic engineering methods. Forexample, 14-hydroxy-11,17-eicosadienoic acid, which is present in someLesquerella species (Smith 1985), is thought to be produced bydesaturation of 14-hydroxy-11-eicosenoic acid.

Again, not only can sequences such as shown in FIG. 10 be used toidentify homologous plant fatty acyl hydroxylases, but the resultingsequences obtained therefrom may also provide a further method to obtainplant fatty acyl hydroxylases from other plant sources. In particular,PCR may be a useful technique to obtain related plant fatty acylhydroxylases from sequence data provided herein. One skilled in the artwill be able to design oligonucleotide probes based upon sequencecomparisons or regions of typically highly conserved sequence. Ofspecial interest are polymerase chain reaction primers based on theconserved regions of amino acid sequence between the castor oleatehydroxylase and the Arabidopsis fad2 shown in FIG. 11. Details relatingto the design and methods for a PCR reaction using these probes isdescribed more fully in the examples.

It should also be noted that the fatty acyl hydroxylases of a variety ofsources can be used to investigate fatty acid hydroxylation events in awide variety of plant and in vivo applications. Because all plantsappear to synthesize fatty acids via a common metabolic pathway, thestudy and/or application of one plant fatty acid hydroxylase to aheterologous plant host may be readily achieved in a variety of species.

Once the nucleic acid sequence is obtained, the transcription, ortranscription and translation (expression), of the plant fatty acylhydroxylases in a host cell is desired to produce a ready source of theenzyme and/or modify the composition of fatty acids found therein in theform of free fatty acids, esters (particularly esterified toglycerolipids or as components of wax esters) or ethers. Other usefulapplications may be found when the host cell is a plant host cell, invitro and in vivo.

For example, by increasing the amount of an oleate hydroxylase availableto the plant, an increased percentage of ricinoleate or lesqueroleate(14-hydroxy-11-eicosenoic acid) may be provided.

Oleate Hydroxylase

By this invention, a mechanism for the biosynthesis of ricinoleic acidin plants is demonstrated. Namely, that a specific plant oleatehydroxylase having preferential activity toward fatty acyl substrates isinvolved in the accumulation of hydroxylated fatty acids in at leastsome plant species. The use of the terms ricinoleate or ricinoleic acidis intended to include the free acids, the ACP and CoA esters, the saltsof these acids, the glycerolipid esters (particularly thetriacylglycerol esters), the wax esters, and the ether derivatives ofthese acids.

The determination that plant fatty acyl hydroxylases are active in thein vivo production of hydroxylated fatty acids suggests severalpossibilities for plant enzyme sources. Hydroxylated fatty acids arefound in some natural plant species in abundance. For example, threehydroxy fatty acids related to ricinoleate occur in major amounts inseed oils from various Lesquerella species. Of particular interest,Lesquerolic acid is a 20 carbon homolog of ricinoleate with twoadditional carbons at the carboxyl end of the chain (Smith 1985). Othernatural plant sources of hydroxylated fatty acids are seeds of the Linumgenus (van de Loo et al., 1993), seeds of Wrightia species, Lycopodiumspecies, Strophanthus species, Convolvulaces species, Calendula speciesand many others (Gunstone et al., 1986).

Plants having significant presence of ricinoleate are preferredcandidates to obtain naturally-derived oleate hydroxylases. However, itwill also be recognized that other plant sources which do not have asignificant presence of ricinoleate may be readily screened as otherenzyme sources. For example, Lesquerella densipila contains adiunsaturated 18 carbon fatty acid with a hydroxyl group (Gunstone etal., 1986) that is thought to be produced by an enzyme that is closelyrelated to the castor oleate hydroxylase, according to the theory onwhich this invention is based. In addition, a comparison between oleate-preferring plant fatty acyl hydroxylases and between plant fatty acylhydroxylases which introduce hydroxyl groups at positions other than the12-carbon or on substrates other than oleic acid may yield insights forprotein modeling or other modifications to create synthetic hydroxylasesas discussed above. For example, on the basis of information gained fromstructural comparisons of the Δ12 desaturases and the oleatehydroxylase, genetic modifications may be made in the structural genesfor Δ15 desaturases that convert these desaturases to 15-hydroxylases(on 18 carbon fatty acids). Since the difference between a hydroxylaseand a desaturase concerns the disposition of one proton, it iscontemplated that by systematically changing the charged groups in theregion of the enzyme near the active site, this change can be effected.

Especially of interest are fatty acyl hydroxylases which demonstrateactivity toward fatty acyl substrates other than oleate, or whichintroduce the hydroxyl group at a location other than the C12 carbon. Asnoted above, such fatty acids may be obtained by expressing the oleatehydroxylase gene in plant species such as oilseed rape that containsuitable substrates other than oleate. As described above, other plantsources may also provide sources for these enzymes through the use ofprotein purification, nucleic acid probes, antibody preparations,protein modeling, or sequence comparisons, for example, and of specialinterest are the respective amino acid and nucleic acid sequencescorresponding to such plant fatty acyl hydroxylases. Also as previouslydescribed, once nucleic acid sequence is obtained for the given planthydroxylase, further plant sequences may be compared and/or probed toobtain homologously related DNA sequences thereto and so on.

Genetic Engineering Applications

As is well known in the art, once a cDNA clone encoding a plant oleatehydroxylase is obtained, it may be used to obtain its correspondinggenomic nucleic acid sequences.

The nucleic acid sequences which encode plant fatty acyl hydroxylasesmay be used in various constructs, for example, as probes to obtainfurther sequences from the same or other species. Alternatively, thesesequences may be used in conjunction with appropriate regulatorysequences to increase levels of the respective hydroxylase of interestin a host cell for the production of hydroxylated fatty acids or studyof the enzyme in vitro or in vivo or to decrease or increase levels ofthe respective hydroxylase of interest for some applications when thehost cell is a plant entity, including plant cells, plant parts(including but not limited to seeds, cuttings or tissues) and plants.

A nucleic acid sequence encoding a plant oleate hydroxylase of thisinvention may include genomic, cDNA or mRNA sequence. By "encoding" ismeant that the sequence corresponds to a particular amino acid sequenceeither in a sense or anti-sense orientation. By "extrachromosomal" ismeant that the sequence is outside of the plant genome of which it isnaturally associated. By "recombinant" is meant that the sequencecontains a genetically engineered modification through manipulation viamutagenesis, restriction enzymes, and the like. A cDNA sequence may ormay not encode pre-processing sequences, such as transit or signalpeptide sequences. Transit or signal peptide sequences facilitate thedelivery of the protein to a given organelle and are frequently cleavedfrom the polypeptide upon entry into the organelle, releasing the"mature" sequence. The use of the precursor DNA sequence is preferred inplant cell expression cassettes.

Furthermore, as discussed above, the complete genomic sequence of theplant oleate hydroxylase may be obtained by the screening of a genomiclibrary with a probe, such as a cDNA probe, and isolating thosesequences which regulate expression in seed tissue. In this manner, thetranscription and translation initiation regions, introns, and/ortranscript termination regions of the plant oleate hydroxylase may beobtained for use in a variety of DNA constructs, with or without theoleate hydroxylase structural gene. Thus, nucleic acid sequencescorresponding to the plant oleate hydroxylase of this invention may alsoprovide signal sequences useful to direct transport into an organelle 5'upstream non-coding regulatory regions (promoters) having useful tissueand timing profiles, 3' downstream non-coding regulatory region usefulas transcriptional and translational regulatory regions and may lendinsight into other features of the gene.

Once the desired plant oleate hydroxylase nucleic acid sequence isobtained, it may be manipulated in a variety of ways. Where the sequenceinvolves non-coding flanking regions, the flanking regions may besubjected to resection, mutagenesis, etc. Thus, transitions,transversions, deletions, and insertions may be performed on thenaturally occurring sequence. In addition, all or part of the sequencemay be synthesized. In the structural gene, one or more codons may bemodified to provide for a modified amino acid sequence, or one or morecodon mutations may be introduced to provide for a convenientrestriction site or other purpose involved with construction orexpression. The structural gene may be further modified by employingsynthetic adapters, linkers to introduce one or more convenientrestriction sites, or the like.

The nucleic acid or amino acid sequences encoding a plant oleatehydroxylase of this invention may be combined with other non-native, or"heterologous", sequences in a variety of ways. By "heterologous"sequences is meant any sequence which is not naturally found joined tothe plant oleate hydroxylase, including, for example, combination ofnucleic acid sequences from the same plant which are not naturally foundjoined together.

The DNA sequence encoding a plant oleate hydroxylase of this inventionmay be employed in conjunction with all or part of the gene sequencesnormally associated with the oleate hydroxylase. In its component parts,a DNA sequence encoding oleate hydroxylase is combined in a DNAconstruct having, in the 5' to 3' direction of transcription, atranscription initiation control region capable of promotingtranscription and translation in a host cell, the DNA sequence encodingplant oleate hydroxylase and a transcription and translation terminationregion.

Potential host cells include both prokaryotic and eukaryotic cells. Ahost cell may be unicellular or found in a multicellular differentiatedor undifferentiated organism depending upon the intended use. Cells ofthis invention may be distinguished by having a plant oleate hydroxylaseforeign to the wild-type cell present therein, for example, by having arecombinant nucleic acid construct encoding a plant oleate hydroxylasetherein.

Depending upon the host, the regulatory regions will vary, includingregions from viral, plasmid or chromosomal genes, or the like. Forexpression in prokaryotic or eukaryotic microorganisms, particularlyunicellular hosts, a wide variety of constitutive or regulatablepromoters may be employed. Expression in a microorganism can provide aready source of the plant enzyme. Among transcriptional initiationregions which have been described are regions from bacterial and yeasthosts, such as E. coli, B. subtilis, Saccharomyces cerevisiae, includinggenes such as beta-galactosidase, T7 polymerase, tryptophan E and thelike.

For the most part, the constructs will involve regulatory regionsfunctional in plants which provide for modified production of plantoleate hydroxylase with resulting modification of the fatty acidcomposition. The open reading frame, coding for the plant oleatehydroxylase or functional fragment thereof will be joined at its 5' endto a transcription initiation regulatory region such as the wild-typesequence naturally found 5' upstream to the oleate hydroxylasestructural gene. Numerous other transcription initiation regions areavailable which provide for a wide variety of constitutive orregulatable, e.g., inducible, transcription of the structural genefunctions. Among transcriptional initiation regions used for plants aresuch regions associated with the structural genes such as for nopalineand mannopine synthases, or with napin, soybean β-conglycinin, oleosin,12S storage protein, the cauliflower mosaic virus 35S promoters and thelike. The transcription/translation initiation regions corresponding tosuch structural genes are found immediately 5' upstream to therespective start codohs. In embodiments wherein the expression of theoleate hydroxylase protein is desired in a plant host, the use of all orpart of the complete plant oleate hydroxylase gene is desired; namelyall or part of the 5' upstream non-coding regions (promoter) togetherwith the structural gene sequence and 3' downstream non-coding regionsmay be employed. If a different promoter is desired, such as a promoternative to the plant host of interest or a modified promoter, i.e.,having transcription initiation regions derived from one gene source andtranslation initiation regions derived from a different gene source,including the sequence encoding the plant oleate hydroxylase ofinterest, or enhanced promoters, such as double 35S CaMV promoters, thesequences may be joined together using standard techniques.

For such applications when 5' upstream non-coding regions are obtainedfrom other genes regulated during seed maturation, those preferentiallyexpressed in plant embryo tissue, such as transcription initiationcontrol regions from the B. napus napin gene, or the Arabidopsis 12Sstorage protein, or soybean β-conglycinin (Bray et al., 1987), aredesired. Transcription initiation regions which are preferentiallyexpressed in seed tissue, i.e., which are undetectable in other plantparts, are considered desirable for fatty acid modifications in order tominimize any disruptive or adverse effects of the gene product.

Regulatory transcript termination regions may be provided in DNAconstructs of this invention as well. Transcript termination regions maybe provided by the DNA sequence encoding the plant oleate hydroxylase ora convenient transcription termination region derived from a differentgene source, for example, the transcript termination region which isnaturally associated with the transcript initiation region. Where thetranscript termination region is from a different gene source, it willcontain at least about 0.5 kb, preferably about 1-3 kb of sequence 3' tothe structural gene from which the termination region is derived.

Plant expression or transcription constructs having a plant oleatehydroxylase as the DNA sequence of interest for increased or decreasedexpression thereof may be employed with a wide variety of plant life,particularly, plant life involved in the production of vegetable oilsfor edible and industrial uses. Most especially preferred are temperateoilseed crops. Plants of interest include, but are not limited torapeseed (Canola and high erucic acid varieties), flax, sunflower,safflower, cotton, Cuphea, soybean, peanut, coconut and oil palms andcorn. Depending on the method for introducing the recombinant constructsinto the host cell, other DNA sequences may be required. Importantly,this invention is applicable to dicotyledons and monocotyledons speciesalike and will be readily applicable to new and/or improvedtransformation and regulation techniques.

The method of transformation is not critical to the current invention;various methods of plant transformation are currently available. Asnewer methods are available to transform crops, they may be directlyapplied hereunder. For example, many plant species naturally susceptibleto Agrobacterium infection may be successfully transformed viatripartite or binary vector methods of Agrobacterium mediatedtransformation. In addition, techniques of microinjection, DNA particlebombardment, electroporation have been developed which allow for thetransformation of various monocot and dicot plant species.

In developing the DNA construct, the various components of the constructor fragments thereof will normally be inserted into a convenient cloningvector which is capable of replication in a bacterial host, e.g., E.coli. Numerous vectors exist that have been described in the literature.After each cloning, the plasmid may be isolated and subjected to furthermanipulation, such as restriction, insertion of new fragments, ligation,deletion, insertion, resection, etc., so as to tailor the components ofthe desired sequence. Once the construct has been completed, it may thenbe transferred to an appropriate vector for further manipulation inaccordance with the manner of transformation of the host cell.

Normally, included with the DNA construct will be a structural genehaving the necessary regulatory regions for expression in a host andproviding for selection of transformant cells. The gene may provide forresistance to a cytotoxic agent, e.g., antibiotic, heavy metal, toxin,etc., complementation providing prototropy to an auxotrophic host, viralimmunity or the like. Depending upon the number of different hostspecies the expression construct or components thereof are introduced,one or more markers may be employed, where different conditions forselection are used for the different hosts.

It is noted that the degeneracy of the DNA code provides that some codonsubstitutions are permissible of DNA sequences without any correspondingmodification of the amino acid sequence.

As mentioned above, the manner in which the DNA construct is introducedinto the plant host is not critical to this invention. Any method whichprovides for efficient transformation may be employed. Various methodsfor plant cell transformation include the use of Ti- or Ri-plasmids,microinjection, electroporation, infiltration, imbibition, DNA particlebombardment, liposome fusion, DNA bombardment or the like. In manyinstances, it will be desirable to have the construct bordered on one orboth sides of the T-DNA, particularly having the left and right borders,more particularly the right border. This is particularly useful when theconstruct uses A. tumefaciens or A. rhizogenes as a mode fortransformation, although the T-DNA borders may find use with other modesof transformation.

Where Agrobacterium is used for plant cell transformation, a vector maybe used which may be introduced into the Agrobacterium host forhomologous recombination with T-DNA or the Ti- or Ri-plasmid present inthe Agrobacterium host. The Ti- or Ri-plasmid containing the T-DNA forrecombination may be armed (capable of causing gall formation) ordisarmed (incapable of causing gall), the latter being permissible, solong as the vir genes are present in the transformed Agrobacterium host.The armed plasmid can give a mixture of normal plant cells and gall.

In some instances where Agrobacterium is used as the vehicle fortransforming plant cells, the expression construct bordered by the T-DNAborder(s) will be inserted into a broad host spectrum vector, therebeing broad host spectrum vectors described in the literature. Commonlyused is pRK2 or derivatives thereof. See, for example, Ditta et al.,(1980), which are incorporated herein by reference. Included with theexpression construct and the T-DNA will be one or more markers, whichallow for selection of transformed Agrobacterium and transformed plantcells. A number of markers have been developed for use with plant cells,such as resistance to kanamycin, the aminoglycoside G418, hygromycin, orthe like. The particular marker employed is not essential to thisinvention, one or another marker being preferred depending on theparticular host and the manner of construction.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime for transformation, the bacteria killed, and the plant cellscultured in an appropriate selective medium. Once callus forms, shootformation can be encouraged by employing the appropriate plant hormonesin accordance with known methods and the shoots transferred to rootingmedium for regeneration of plants. The plants may then be grown to seedand the seed used to establish repetitive generations and for isolationof vegetable oils.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES

In the experimental disclosure which follows, all temperatures are givenin degrees centigrade (°), weights are given in grams (g), milligram(mg) or micrograms (μg), concentrations are given as molar (M),millimolar (mM) or micromolar (μM) and all volumes are given in liters(1), microliters (μl) or milliliters (ml), unless otherwise indicated.

Isolation of Castor Oleate Hydroxylase cDNA Overview

Ricinoleic acid is specific to the seed tissue of castor, and is notfound in vegetative tissues (Canvin 1963; James et al., 1965).Therefore, a differential screening approach was used to enrich for cDNAclones which were expressed in seeds but not in leaves. A large numberof clones with these properties were retained and partial nucleotidesequence information was obtained from each clone. The nucleotidesequences were translated in all six possible reading frames and thededuced amino acids sequences were compared to the sequences of plantfatty acid desaturases in order to identify clones which exhibited aminoacid sequence homology. Candidate clones were then placed undertranscriptional control of a plant promoter and introduced intotransgenic plants of tobacco and Arabidopsis thaliana. Finally, thepresence of ricinoleic acid in the seed oils of these transgenic plantswas verified by gas chromatography and mass spectrometry. The varioussteps involved in this process are described in detail below.

RNA Isolation

Total RNA was purified from developing stage III to stage V (Greenwoodand Bewley, 1982) castor cellular endosperm plus embryo by the techniqueof Puissant and Houdebine (1990) with minor modifications. Briefly,tissue (10 g) was powdered in liquid nitrogen and divided into 8 tubes.The frozen powder was suspended in 5 ml buffer (4M guanidiniumthiocyanate, 25 mM sodium citrate pH 7.0, 0.5 % sarkosyl, 0.1M2-mercaptoethanol). The following reagents were added, punctuated byvortexing of the tube: 2M sodium acetate pH 4.0 (0.5 ml), phenol (5 ml),and chloroform (1.0 ml). Following incubation on ice for 15 min, thetubes were centrifuged at 10,000 g (7000 rpm) for 10 min. Isopropanol (5ml) was added to the upper phase and incubated on ice for 10 min,followed by centrifugation as before. The RNA pellet was dislodged with1 ml 4 M LiCl and transferred to a microfuge tube. The original tube wasrinsed with 0.5 ml more LiCl and the pellet vortexed for 5 min in thecombined liquid. RNA was pelleted in a microfuge (10 min), thenresuspended again in 1 ml 4M LiCl and pelleted again. The pellet wasthoroughly resuspended in TE/0.5% SDS (750 μl) and extracted with anequal volume of chloroform/isoamyl alcohol (24:1). The aqueous phase wasextracted a second time before precipitation of RNA by the addition of2M sodium acetate (100 μl) and isopropanol (600 ul). RNA was pelletedand resuspended in water, and represented the purified total RNAfraction.

Production of cDNA Libraries

PolyA⁺ RNA (10ug) was prepared from total castor RNA (1.5 mg) by twopasses down an oligo (dT) spin column. This was done using a kit(catalog number 5302-600750) according to the instructions of themanufacturer (5 Prime--3 Prime, Inc. 5603 Arapahoe, Boulder, Colo. 80303USA).

A λZAPII cDNA library was prepared using a ZAP-cDNA synthesis kit(Stratagene, 11011 North Torrey Pines Road, La Jolla, Calif. 92037.catalog number 200400). First and second strand cDNA was synthesizedfrom polyA⁺ RNA (5 μg) using an oligo (dT) primer and Moloney-MurineLeukemia Virus Reverse Transcriptase exactly as described by themanufacturers instructions. Following addition of EcoRI linkers anddigestion with XhoI the cDNA was purified on a sephacryl S-400 (Sigmachemical Company, PO box 14508, St Louis, Mo. 63178, USA. Catalog numberS-400-HR) spin column prepared according to the instructions in theZAP-cDNA synthesis kit. The cDNA was loaded onto the column which hadbeen equilibrated in 10 mM Tris-Cl (pH 8.0), 1 mMEDTA. cDNA (400 ng)eluting in the second fraction was concentrated by ethanolprecipitation. Half of this cDNA (200 ng) was ligated into Lambda ZAPIIdigested with XhoI and EcoRI according to the instructions supplied withthe ZAP-cDNA synthesis kit. Construction of the λZAPII library includeddirectional cloning, so that 5' ends of the inserts should be found atthe T3 side of the polylinker. The entire ligation was packaged usingGigapack packaging extract (Stratagene, 11011 North Torrey Pines Road,La Jolla, Calif. 92037. catalog number 200211) according to themanufacturers instruction and plated on E. coli strain XL1-Blue(Stratagene, 11011 North Torrey Pines Road, La Jolla, Calif.). Thisyielded 1×10⁵ primary plaques which were eluted in SM buffer (100 mMNaCl, 8 mM MgSO₄ 50 mM Tris-HCl pH 7.5, 0.1% gelatin per liter) andstored at 4° C.

A second cDNA library was prepared in the plasmid vector pYES2(Invitrogen). Complementary DNA was prepared using a kit ("LibrarianIV", Invitrogen) according to the instructions of the manufacturer.First strand cDNA (1.65 μg) was synthesized from poly(A)⁺ RNA (5 μg) bypriming with oligo dT and extension by avian myeloma virus reversetranscriptase. The RNA was nicked by E. coli RNaseH, forming primers forsecond-strand cDNA synthesis by E. coli DNA polymerase I. Any nicks inthe dsDNA were repaired with E. coli DNA ligase. Ends of the dsDNA weremade blunt with T4 DNA polymerase for ligation of BstX1 non-palindromiclinkers. The cDNA was size-selected by agarose-gel electrophoresis, andmolecules larger than ˜750 bp were ligated into the BstXl-digestedpYES2.0 vector and transformed into E. coli strain INV1αF', yieldingfour pools containing a total of 1.42×10⁶ transformants.

Differential Screening of λZAPII Library

Phage from the castor λZAPII library was picked randomly into eighteenseparate 96-well plates (designated #1-9 and #28-36). These werereplicated onto bacterial lawns prepared by adding 0.2 ml of a saturatedL-broth culture of E. coli strain XL1-blue (Stratagene) to 5 ml ofmolten top agar and pouring the mixture onto the surface ofagar-solidified L-broth medium in a 132 mm petri dish. Each 96-wellplate was replicated using a 96-prong device which could be lowered ontothe lawn through a 3×3 array of guides. The blunt ˜1 mm diameter prongscarried sufficient phage to give plaques of consistent size, withoutsignificant encroachment between neighboring plaques. Multiple filters,each representing 864 identifiable clones, were lifted from theresulting plaques and screened with ³² P-labelled first-strand cDNAprobes reverse transcribed from leaf or developing endosperm/embryopoly(A)⁺ RNA. Triplicate nylon filters (Hybond N⁺, Amersham) were liftedfrom these plaques. DNA was fixed to the filters by placing them onfilter paper moist with denaturing solution (0.5M NaOH, 1.5M NaCl; 5min), neutralizing solution (0.5M Tris-Cl pH 7.4, 1.5M NaCl; 5 min), and2×SSC (0.3M NaCl, 0.03M Na-Citrate, pH 7.0). The filters were thenair-dried, with no further fixation of the DNA. The filters werescreened with the various probes described below. For plates 1-9,polyadenylic acid (1 μg ml⁻¹) was added to the hybridization solution,and results for plates 1-9 were obtained from a phosphor-imager(Molecular Dynamics) rather than from autoradiographs. Exposure timeswere: plates 1-9, 21 h (note that phosphor imaging is several-fold moresensitive than autoradiography); plates 28-36 leaf probe 3 days, seedprobe 24 h, redundant-clone probe 1.5 h.

Probes for differential screening plates 1-9 were prepared as follows.Poly(A)⁺ RNA (1 μg) from seed or leaf in a volume of 17 μl was heated to70° C. for 5 min, then chilled in ice-water, and added to the reactiontube, to a final volume of 50 μl. The reaction mixture contained inaddition: 50 U RNasin (Promega), 1× reverse transcriptase buffer(Boehringer-Mannheim), 20 ng/μl oligo(dT)₁₂₋₁₈, 1 mM each of dGTP, dATP,dTTP, 4.8 μM dCTP (unlabelled), 100 μCi α-³² P dCTP (3000 Ci mmol⁻¹), 40U Avian Myeloma Virus Reverse Transcriptase. The reaction was incubatedat 42° C. for 60 min. The reaction was stopped and RNA removed byaddition of EDTA (to 16 mM), SDS (to 0.4%), NaOH (to 0.4M) andincubation at 65° C. for 30 min. The probe was neutralized with 6 μl 2MHCl and 20 μl 1M Tris-Cl, pH7.4, then precipitated with 375 μl EtOH inthe presence of 0.7M ammonium acetate and 10 μg denatured carrier(salmon sperm) DNA. After incubation at -20° C. for ˜3 h, DNA (˜60% oftotal radioactivity) was pelleted by centrifugation for 15 min, andresuspended in 200 μl water and added to the filters.

For plates 28-36, first-strand cDNA was made using the same RNA (0.5 μgseed, 1.2 μg leaf) in a reverse transcription reaction similar to thatdescribed above, but using unlabelled nucleotides and all othercomponents from a reverse transcription kit (Promega). The RNA washydrolysed and the cDNA was neutralized as described above, and thenpurified by batch chromatography on glass (GeneClean, Bio101). The cDNAwas then labelled by random hexamer priming using 100 μCi α³² P dCTP.The probes were precipitated as described above, and heated to 100° C.(5 min) before addition to the filters. Incorporation of radioactivitywas ˜60% (leaf probe) or ˜30% (seed probe).

Only those clones were selected which gave no detectable signal with theprobe derived from leaf mRNA, and did not give a very strong signal withthe probe derived from seed mRNA. Plates 1˜9 were processed in thismanner, from which the first batch of cDNA sequences were obtained(described below).

Of 864 possible plaques from plates 1-9, 10 did not develop and 15 wereoccluded by bubbles separating the plaque and filter, leaving 839 cloneswith DNA on the filter. Of these, 162 (19.3%) were scored as having astrong seed signal, while 280 (33.4%) gave no detectable signal with theleaf probe. Of these 280, 222 were not among the previous category andwere selected for sequencing. These results therefore indicated that 222of 839, or 26.5% of clones, were in the category "seed-specific and nothighly abundant". Of the 162 clones having a strong seed signal, only 58appeared to be seed specific.

Some changes were made when screening plates 28-36 for the secondsequencing batch. The seed mRNA and leaf mRNA probes were made by randompriming using first-strand cDNA as a template, in an attempt to gainmaximum incorporation of radioactivity into less-abundant sequences. Inaddition, a probe was made from the pooled insert DNA of clones thatwere sequenced several times in the first batch so that fewer redundantsequences would be obtained. A mixed probe was made from some of themost redundant clones as follows. Plasmid DNA of highly representedclones (Table 1) were digested with BamHI and KpnI and the insertspurified from agarose gels. DNA of these inserts was pooled and ˜600 nglabelled with 100 μCi α³² P dCTP (˜80% incorporation) by random primingas described above. Screening results were obtained directly fromautoradiograms. An example of the autoradiograms is presented in FIGS.1-3.

For plates 28-36, 851 of a possible 864 plaques were represented on thefilter, and of these 851, 370 (43.5%) gave a strong seed signal, 512(60.2%) gave no detectable leaf signal, and 141 (16.6%) gave a signalwith the probe made from redundant sequences (the effectiveness ofscreening with this particular probe is discussed below). This resultedin the selection of 348 (40.9% of 851) clones to be sequenced.

TABLE 1

List of highly expressed castor cDNA clones of known function fromplates 1-9.

Ribosomal proteins: Clones pCRS262, pCRS312, pCRS356, pCRS358, pCRS377,pCRS396, pCRS407, pCRS409, pCRS416, pCRS426, pCRS432, pCRS442, pCRS446.

12S seed storage protein: Clones pCRS267, pCRS269, pCRS298, pCRS404,pCRS405, pCRS408, pCRS434, pCRS443, pCRS453, pCRS454.

2S seed storage protein: Clones pCRS281, pCRS328, pCRS337, pCRS362,pCRS375, pCRS431.

Meat shock proteins: Clones pCRS264, pCRS348, pCRS397.

Enolase: Clones pCRS330, pCRS380, pCRS415, pCRS439.

DNA Sequencing

The differential screens described above gave a total of 570 lambdaphage clones selected for sequencing. The phage were converted toplasmids by a slightly modified (scaled-down) version of the Stratageneprotocol provided with the purchase of the λZAP cDNA synthesis kit.Briefly, in a 15 ml conical tube, combine 200 μl XL1-Blue cells, 20 μlphage suspension from 96-well plate, and 1 μl R408 helper phage(Stratagene). Incubate at 37° C. for 15 min, then add 5 ml 2×YT medium(per liter: 10 g NaCl, 10 g yeast extract, 16 g bacto-tryptone), andshake at 37° C. for 3 h. Heat to 70° C. for 20 min, centrifuge 5 min at4000 g, and store supernatant (phagemid stock) at 4° C. To obtaincolonies, mix 200 μl XL1-Blue cells and 1 μl of 1/100 dilution ofphagemid stock, incubate 15 min at 37° C. plate 100 μl on LB agar mediumcontaining 100 μg/ml ampicillin and incubate at 37° C. until coloniesform (ca. 18 h).

Plasmid DNA was prepared from E. coli cultures (5 ml, LB mediumcontaining 100 mg 1⁻¹ ampicillin) using "Magic Minipreps" (Promega)according to the instructions of the manufacturer. DNA was analyzedspectrophotometrically for DNA concentration, and submitted to theMichigan State University, Plant Research Laboratory Sequencing Facilityfor automated sequencing on Applied Biosystems 373A DNA sequenators. TheT3 primer (Applied Biosystems, Foster City, Calif.) was generally usedto prime the sequencing reactions. Sequence data was manually edited toremove vector/linker sequences, and truncated at the point wheresequence quality declined substantially as indicated by a highproportion of ambiguous nucleotide identifications. These editedsequences (typically 400-500 nucleotides) were compared with the publicsequence databases by electronic submission of the sequence to the BLASTserver (BLAST@ncbi.nlm.nih.gov) provided by the National Center forBiotechnology Information, Bethesda, Md. DNA sequences were compared inall reading frames to the non-redundant translated-nucleotide andprotein sequence databases (Swiss-Prot 24.0 or 25.0 plus weekly updates;PIR 35.0, 36.0, or 37.0; GenBank Release 75.0, 76.0, or 77.0, plus dailyupdates; and EMBL Release 34.0 or 35.0, plus daily updates) by theprogram blastx (Altschul et al.,1990) in the months March-July, 1993.

Of the 526 clones sequenced, 58 gave sequence data which was notconsidered informative because of poor quality or the presence of onlyvector sequences. Sequence from the 468 informative clones was analyzedby the blastx program, leading to the putative identification of 213(46%) of them by the criteria that these partial sequences had blastxscores greater than 80. DNA sequences generated in this study have beendeposited in the NCBI database, dbEST (database for Expressed SequenceTags), as identification numbers 39704-40169, and in GenBank, asaccession numbers T14820-T15266. The sequences will not be made publiclyaccessible until after the filing date of this patent application.

Two clones pCRS677 (dbEST accession number 40094) and pCRS834 (dbESTaccession number 40142) have sequence similarity with plantmembrane-bound desaturase genes. The original uncorrected partialnucleotide sequences for pCRS677 and pCRS834 on which this conclusionwas based are shown in FIGS. 4 and 5, respectively. The homology isshown in FIGS. 6A and 6B where the deduced amino acid sequence oftranslation frame +2 obtained from clone pCRS677 is compared to thededuced amino acid sequence of the microsomal ω3 fatty acyl desaturasefrom Brassica napus and a cDNA from Vigna radiata that is also thoughtto be a fatty acid desaturase (Iba et al., 1993). A similar result isshown in FIGS. 7A and 7B for clone pCRS834. Therefore these clones wereselected for further analysis as putative clones of the oleatehydroxylase.

Isolation and Sequencing of cDNA Clone pFL2

Comparison of the initial partial (uncorrected) sequence data of pCRS677and pCRS834 obtained with the T3 primer (FIGS. 4 and 5), indicated thatthese are probably independent clones derived from the same gene (FIGS.8A and 8B). Although there are a number of differences between the twonucleotide sequences, these are mostly located at the 3' end of thesequences and are, therefore, thought to be sequencing errors resultingfrom the inaccuracy of the base-calling routines of the automatedsequenator used to obtain these partial sequences.

The insert of pCRS677 (˜700 bp) was used as a probe to screen a castorpYES2.0 library by colony hybridization at high stringency. Three 100 mmplates of each of the four pools of the pYES2.0 cDNA library werescreened by the same methods described above. In brief, E. coli cellscontaining the pYES2.0 library were plated at a density of approximately39,000 colonies per 100 mm petri dish on agar solidified LB mediumcontaining ampicillin (100 μg/ml) and grown at 37° C. until smallcolonies were visible. A nitrocellulose filter (Schleicher & SchellBAS5) was laid on each plate, its position marked, and lifted off to afresh plate, the adhering colonies now facing upwards. Care wasnecessary that both plate and filter were not too moist, to avoidsmearing of the colonies. The original plate was incubated for 5 h at37° C. to recover colonies, while the filters were processed as follows.Each filter was sequentially placed, colony side up, on Whatman 3 MMpaper moist with 10% SDS (3 min), denaturing solution (0.5M NaOH, 1.5 MNaCl; 5 min), neutralizing solution (0.5M Tris-Cl pH 7.4, 1.5M NaCl; 5min), and 2×SSC (0.3M NaCl, 0.03M Na-Citrate, pH 7.0). The filters werethen air-dried for ca. 1 min before pressing twice between sheets offilter paper to remove cell debris. After air-drying a further 30 min,DNA was fixed to the filters by baking in vacuo at 80° C. for 1-2 h.

The filters were prehybridised in a minimal volume of 4×SET (0.6M NaCl,0.12M Tris-Cl pH 7.4, 8 mM EDTA), 0.1% Na-pyrophosphate, 0.2% SDS, 100μg/ml heparin, at 65° C., before addition of the probe and hybridizationovernight. The pCRS677 insert was excised with BamHI and ApaI,gel-purified, ³² P-labelled by random priming and purified ofunincorporated nucleotides by ethanol precipitation in the presence ofammonium acetate. This probe was hybridized to the filters overnight at65° C. The filters were washed three times in 2×SSC, 0.1% SDS at 65° C.,then exposed to X-ray film.

In the primary screen of 47,000 colonies, 84 hybridizing colonies wereobtained. The first 28 of these positive colonies were purified bystreaking for single colonies. All 28 of the primary positives werepositive in the secondary screen, indicating an overall frequency of onepositive clone per 560 clones in the cDNA library. DNA prepared from the28 purified clones was digested with restriction enzymes and analyzed byagarose gel electrophoresis. The enzymes BamHI and XhoI cut the vectoron either side of the cloning site, and therefore should excise theinserted DNA when used together. With one exception, all clones had asingle fragment smaller than ˜800 bp, or an ˜800 bp fragment plus one ortwo additional fragments. Clone 4avi did not fit this pattern. Adouble-digest with XbaI and HindIII should, similarly, excise theinsert. All clones analyzed yielded only one fragment, ranging in sizebetween ˜700 bp and ˜2.2 kb, except clone 4avi, which had an insert of˜4 kb. Due to minor technical difficulties, however, clones 2ci, 3cv,4cii, 4aiii, 4ci, 4aii, 4ai, and 3ciii, were not analyzed by digestionwith XbaI and HindIII. The majority of clones had one HincII site in theinsert, with the exception of clones 3cv, 4cii, 4aiii, 4ci, 3cii, 4aii,3cvii, and 3cvi, which either lacked this site or had an additionalsite. Taken together, these results indicate that most of the 28 clonespurified had a similar restriction pattern, with 9 possible exceptions.This indicated that most, if not all, represent the same gene. Of themajority of clones, which appeared to have similar restriction patternsbut varying insert sizes, 10 were used to obtain partial sequence datashown in FIG. 9. This sequence data indicated that these 10 clones hadhighly similar sequences and were probably derived from the same gene.It is concluded that this one class of clones is present in the pYES2.0library at a frequency between 1/560 and 1/1120. The longest clone,3cvii, was 113 bp longer than the next longest, 3civ-1. However, thefirst 305 bp of 3cvii showed no similarity to the overlapping portion of3civ-1 or several other clones of similar length, which were, however,all highly similar in sequence to each other (FIG. 9). It was concludedthat the first 305 bp of the cDNA in clone 3cvii contained extraneousDNA, not related to pCRS677 (nor any other known sequence). Furthersequence data was obtained only from clone 3civ-1, hereafter designatedpFL2. The gene corresponding to the insert in pFL2 is hereafterdesignated by the symbol fah12 (fatty acid 12-hydroxylase).

DNA Sequencinq

Nucleotide sequences of cloned DNA fragments can be obtained by avariety of commonly used methods. DNA sequencing of the DNA fragmentsdescribed herein was performed with an ABI Catalyst-8000 robot and anABI373A DNA sequencer using dye terminator or dye primer sequencingreactions. Sequence data was analyzed using the programs DNASIS andPROSIS (Hitachi Company).

The sequence of the insert in clone pFL2 is shown in FIGS. 10A and 10B.The sequence entails 1448 bp of contiguous DNA sequence (SEQ ID NO:39).The clone encodes a 186 bp 5' untranslated region (i.e. nucleotidespreceding the first ATG codon), an 1161 bp open reading frame, and a 101bp 3' untranslated region, including a short (9 bp) poly(A) tail. Theopen reading frame encodes a 387 amino acid protein with a predictedmolecular weight of 44406.8 (SEQ ID NO: 40). The amino terminus lacksfeatures of a typical signal peptide (yon Heijne, 1985). The predictedsequence of the Brassica napus fad3 microsomal desaturase also lacks atypical signal peptide (Arondel et al., 1992).

The exact translation-initiation methionine has not been experimentallydetermined, but on the basis of deduced amino acid sequence homology tothe microsomal ω6 fatty acyl desaturase (noted below) is thought to bethe methionine encoded by the first ATG codon at nucleotide 187.

Comparison of the pFL2 nucleotide and deduced amino acid sequences withsequences of membrane-bound desaturases (Table 2) indicates that pFL2 ishomologous to these genes. Sequence similarity between pFL2 and thesedesaturase genes is considerably weaker than similarities among thedesaturase genes. An alignment of the deduced amino acid sequences ofthe insert in pFL2 and the Arabidopsis fad2 cDNA which encodes anendoplasmic reticulum-localized ω-6 (Δ12) desaturase (Okuley et al.,1994) is shown in FIG. 11. The overall homology between the two geneproducts was 67% and the length of the sequences differed by only 4amino acid residues. Thus, in view of the fact that the two genes arefrom distantly related plants, the high degree of sequence homologyindicates that the gene products are of similar function.

The deduced amino acid sequence of pFL2 (FIGS. 10A and 10B) contains theconserved histidine-rich repeats (HXXHH) also found in all known plantmembrane-bound desaturases (Arondel et al., 1992; Iba et al., 1993;Yadav et al., 1993; Okuley et al., 1994).

                  TABLE 2                                                         ______________________________________                                        Amino acid (AA) and nucleotide (NT) sequence                                  similarity between the cDNA in pFL2 and                                       membrane-bound desaturase genes                                                              % Identity                                                     Organism    Gene     AA     NT   Function                                     ______________________________________                                        Ricinus communis                                                                          fad7     38.6   47.1 ω3 desaturase                          Brassica napus                                                                            fad3     37.4   46.5 ω3 desaturase                          A. thaliana fad7     35.5   47.4 ω3 desaturase                          A. thaliana fad2     67     65.4 ω6 desaturase                          ______________________________________                                    

Northern Blot Analysis

Ricinoleic acid is generally found only in seed oils andoleate-12-hydroxylase activity is only found in the developing seeds ofcastor. Therefore, an important criterion in discriminating between anω6 fatty acyl desaturase and oleate hydroxylase is that the oleatehydroxylase gene is expected to be expressed more highly in tissueswhich have high level of ricinoleate than in other tissues whereas allplant tissues should contain mRNA for an ω6 fatty acyl desaturase sincediunsaturated fatty acids are found in the lipids of all tissues in mostor all plants. Therefore, it was of great interest to determine whetherpFL2 was also expressed only in seeds, or is also expressed in othertissues. This question was addressed by testing for hybridization ofpFL2 to RNA purified from developing seeds and from leaves.

A northern blot of RNA from leaves and developing seeds from stage IIIto stage V (Greenwood and Bewley 1982) of castor was probed with the ³²P-labelled insert of clone pCRS677, which corresponds to ˜700 bp of the3' end of pFL2.

Poly(A) ⁺ RNA prepared as described above from leaves and developingseeds was electrophoresed through an agarose gel containing formaldehyde(Iba et al., 1993). An equal quantity (3 μg) of RNA was loaded in bothlanes, and RNA standards (0.16-1.77 kb ladder, Gibco-BRL) were loaded ina third lane. Following electrophoresis, RNA was transferred from thegel to a nylon membrane (Hybond N, Amersham) and fixed to the filter byexposure to UV light. A ³² P-labelled probe was prepared from insert DNAof clone pCRS677 as above, and hybridized to the membrane overnight at65° C., after it had been prehybridised for ˜1 h. The hybridizationsolution contained 4×SET (0.6M NaCl, 0.12M Ttis-HCl pH 7.4, 8 mM EDTA),0.1% sodium pyrophosphate, 0.2% SDS, 0.1% heparin, and 5% dextransulphate. The blot was washed lo three times in 2×SSC, 0.1% SDS at roomtemperature, then exposed to X-ray film, and to a phosphor-imagingscreen (Molecular Dynamics). A probe was subsequently made from theColletotrichum graminicola β-tubulin gene TUB2 (Panaccione and Hanau,1990) and hybridized to the same blot under the same conditions, exceptthat the hybridization temperature was reduced to 58 ° C., and wasexposed to X-ray film.

Brief (30 min) exposure of the blot to X-ray film revealed that theprobe hybridized to a single band of ˜1.67 kb, only in the seed RNA lane(FIG. 12, panel A). Upon overexposure (16 h) of the film, a band ofsimilar size was detected in the leaf RNA lane, in addition to a second,larger, band in the seed RNA lane (FIG. 12, panel B). The blot was alsoexposed to a phosphor-imaging screen, for quantitation of probehybridization. Total exposure to this screen in an area coveting theband in the leaf lane was 4.36×10⁴ units above background. Totalexposure in an area of equal size over the major band in the seed lanewas 1.17×10⁷ units above background, 268-fold more than in the leaflane. The blot was re-probed with β-tubulin gene, which gave bands ofequal intensity in the seed and leaf lanes (FIG. 12, panel C), verifyingthat equal quantities of undegraded RNA were loaded in the two lanes.

These results show that the fah12 gene corresponding to the clone pFL2is highly and specifically expressed in seed of castor. Over-exposure ofthe Northern revealed a 268-fold weaker band of similar size in leafRNA, but also a second band in seed RNA, suggesting that these bands aredue to weak hybridization of pFL2 to related sequences, such asmicrosomal ω6 fatty acyl desaturases. In conjunction with knowledge ofthe nucleotide and deduced amino acid sequence, strong seed-specificexpression of pFL2 is a useful indicator of the role of the enzyme insynthesis of hydroxylated fatty acids in the seed oil.

Southern Blot Analysis

Southern analysis was used to examine the copy number of genes in thecastor genome corresponding to clone pFL2, and to examine whetherrelated sequences could be detected in the castor genome, and in thegenome of a different plant, in which oleate-12-hydroxylase is absent.

Genornic Arabidopsis DNA (1 μg) and genomic castor DNA (2 μg) weredigested with EcoRI, BamHI, or HindlII, and separated in 0.7% agarosegel. A Southern blot was prepared as described in (Sambrook et al.,1989). The blot was prehybridised at 65° C. in a solution containing4×SET (0.6M NaCl, 0.12M Tris-HCl pH 7.4, 8 mM EDTA), 0.1% sodiumpyrophosphate, 0.2% SDS, and 100 μg/ml heparin. The probe was hybridizedto the blot at 65° C. overnight in the same solution, except for theaddition of 10% dextran sulphate. The blot was washed three times in2×SSC, 0.1% SDS at room temperature then exposed to X-ray film.Arabidopsis was chosen for the negative control DNA because it has noknown oleate-12-hydroxylase. The membrane was hybridized with the ³²P-labelled insert of clone pFL2 at 65° C., and exposed to X-ray film.

The probe hybridized with a single band in each digest of castor DNA,but did not hybridize to the Arabidopsis DNA (FIG. 13), indicating thatthe fah12 gene from which pFL2 was transcribed is probably present in asingle copy in the castor genome, and is not present in the Arabidopsisgenome. The blot was then hybridized again, with an identical probe, butat less stringent hybridization conditions (52° C.) (FIG. 14). Thisrevealed additional weakly-hybridizing bands in both castor andArabidopsis. In castor DNA, a total of four bands were detected in boththe EcoRI digest and the BamHI digest. In Arabidopsis DNA, four bands(EcoRI), five bands (BamHI), or possibly three bands (HindlII) weredetected. These results suggest that at least one additional gene withsequence similarity to pFL2 occurs in both the castor and Arabidopsisgenomes. Comparison of the nucleotide sequences of the castor fah12 cDNAand the Arabidopsis fad2 cDNA showed several regions of strongnucleotide sequence hornology (FIGS. 15A, 15B and 15C). Thus, some orall of the bands of hybridization observed on low stringency Southernblots are due to hybridization of the fah12 clone to one or more genesfor microsomal ω6 fatty acyl desaturase in both castor and Arabidopsis.

Expression of pFL2 in Transgenic Plants

There are a wide variety of plant promoter sequences which may be usedto cause tissue-specific expression of cloned genes in transgenicplants. For instance the napin promoter and the acyl carrier proteinpromoters have previously been used in the modification of seed oilcomposition by expression of an antisense form of a desaturase (Knutsonet al. 1992). Similarly, the promoter for the β-subunit of soybeanβ-conglycinin has been shown to be highly active and to result intissue-specific expression in transgenic plants of species other thansoybean (Bray et al., 1987). Thus, although we have used the cauliflowermosaic virus 35S promoter in the examples described here, otherpromoters which lead to seed-specific expression are preferred for theproduction of modified seed oil composition. Such modifications of theinvention described here will be obvious to one skilled in the art.

Constructs for expression of castor oleate hydroxylase plant cells whichutilize the CaMV355 promoter region are prepared as follows: The insertof clone pFL2 was ligated between the 35S promoter and nos terminator ofthe plant expression binary vector pBI121 (FIG. 16) (Clontech, PaloAlto, Calif.) in the correct orientation for expression of the openreading frame, by two independent cloning strategies.

The use of the vector pBI121, in which the only 3' cloning site is SacI,was complicated by the presence of a SacI site in the coding region ofthe pFL2 insert. In the first route, pFL2 was linearised with XbaI(which cuts at the 3' region flanking the insert), blunt-ended with theKlenow fragment of DNA polymerase I, then digested with BarnHI (whichcuts at the 5' end of the insert), releasing the insert, which wasgel-purified. The vector pBI121 (FIG. 16) was digested with SacI andblunt-ended with T4 DNA polymerase, then cut with BarnHI and treatedwith calf intestinal phosphatase to prevent religation with the excisedβ-glucuronidase fragment. The pFL2 insert was ligated to this pBI121vector and used to transform Escherichia coli DH5α cells to kanamycinresistance. Plasmid DNA of transformants was digested with XbaI andSacI, and two dories (A4, B6) were chosen that had the 1.3 kb fragmentindicating that the pFL2 cDNA was correctly inserted into the pBI121vector. This was confirmed by the fact that SnaBI did not cut theseclones (SnaBI cuts the β-glucuronidase gene), and EcoRI/HindIII releaseda band of appropriate size (˜2.5 kb).

In the second route, clone pFL2 was digested with XbaI and thenpartially digested with SacI. A band of ˜1.45 kb representing the entireinsert was isolated from a gel. The vector SLJ4K1 (FIG. 17) was obtainedfrom Dr, J. Jones, Sainsbury Institute, John Innes center, Norwich,England. The plasmid was digested with XbaI and SacI, and the vectorfragment was gel-purified. The pFL2 insert was ligated to this vector,transformed into DH5α, and checked for the presence of the 1.3 kb SacIinsert fragment. Such a clone was then digested with EcoRI and HindIII,and this DNA was ligated to the large EcoRI/HindIII fragment of pBI121,transformed into DH5α and selected for both kanamycin resistance andampiciIlin sensitivity. By this procedure, the entire (35Spromoter)-(pFL2 insert)-(nos terminator) fragment derived from SLJ4K1was used to replace the (35S promoter)-(β-glucuronidase)-(nosterminator) fragment of pBI121. The clones obtained were digested withSacI, and one done (9/18 3) which gave the appropriate 1.3 kb fragmentwas selected.

The three clones (A4 and B6 prepared by the first route, and 9/18 3prepared by the second), plus the unmodified vector pBI121, weretransformed into Agrobacterium turaefaciens strains GV3101 and LBA4404by electroporation. GV3101 (Koncz and Schell, 1986) and LBA4404 (Ooms etal., 1982) contain disarmed Ti plasmids. Cells for electropotation wereprepared as follows. GV3101 was grown in LB medium with reduced NaCl (5gl⁻¹), and LBA4404 was grown in TY medium (5 gl⁻¹ bacto-tryptone, 3 gl⁻¹yeast extract, pH 7.5). A 500 ml culture was grown to OD600=0.6, thencentrifuged at 4000 rpm (GS-A rotor) for 5 min. The supernatant wasaspirated immediately from the loose pellet, which was gentlyresuspended in 500 ml ice-cold water. The cells were centrifuged asbefore, resuspended in 30 ml ice-cold water, transferred to a 30 ml tubeand centrifuged at 5000 rpm (SS-34 rotor) for 5 min. This was repeatedthree times, resuspending the cells consecutively in 30 ml ice-coldwater, 30 ml ice-cold 15% dimethyl sulfoxide (DMSO), and finally in 4 mlice-cold 15% DMSO. These cells were allquoted, frozen in liquidnitrogen, and stored at -80° C. Electroporations employed a BTXinstrument using cold 1 mm-gap cuvettes containing 40 μl cells and aminimal volume of DNA, a voltage of 1.44 KV, and 129 Ω resistance. Theelectroporated cells were diluted with 1 ml SOC medium (Sambrook et al.,1989, page A2) and incubated at 28° C. for 1-2 h before plating onmedium containing kanamycin (50 mg l⁻¹.

EXAMPLE 1 Production of Ricinoleate in Transgenic Tobacco

A variety of methods have been developed to insert a DNA sequence ofinterest into the genome of a plant host to obtain the transcription ortranscription and translation of the sequence to effect phenotypicchanges. The following methods represent only one of many equivalentmeans of producing transgenic plants and causing expression of thehydroxylase gene.

Nicotiana tabacura SR-1 leaf explants were transformed according toNewman et al (1993) with minor modifications as noted below. Seeds ofNicotiana tabacum SR-1 are soaked in 95% ethanol for 2 min surfacesterilized in a 1.0% solution of sodium hypochlorite containing a dropof Tween 20 for 45 min, and rinsed three times in sterile, distilledwater. Seeds are then plated in Magenta boxes with 1/10th concentrationof Murashige Skoog (MS) minimal organics medium (Gibco; Grand Island,N.Y.) supplemented with 30 g/L sucrose, 0.56 mM myo-inositol, 2.5 mM MESand adjusted to pH 5.7 and solidified with 0.8% Phytagar (Gibco). Seedsare germinated at 22° C in a 24 h photoperiod with cool fluorescentlight of intensity approximately 50 μEinsteins per square meter persecond (5E m⁻² s⁻¹). Axenic leaf pieces from 3 to 8 week old plants weretransferred to No. 3 medium (MS salts, 30 g/L sucrose, 1.2 μM thiamine,0.56 mM myoinositol, 1 μM indole-3-acetic acid, 10 μM benzylaminopurine,2.5 mM MES and adjusted to ph 5.6 and solidified with 0.65% agar). After3 days of incubation in continuous light of approximately 50 5E m⁻² s⁻¹,the leaf fragments were inoculated by pricking the leaves with sterilesyringe needles dipped in fresh colonies of Agrobacterium. After 3 to 4days the leaf fragments were transferred to No. 3 medium containing 200μg/ml kanamycin and 500 μg/ml carbenicillin. Shoots which emerged duringthe following one to three months were transferred to Magenta boxescontaining 0.65% agar-solidified MS medium containing 1% sucrose, 2 mg/Lindolebutyric acid, 100 μg/ml kanamycin and 500 μg/ml carbenicillin toinduce rooting. Rooted plants were transferred to soil and grown undernatural light in a glasshouse with a mean daily temperature of 28 ° C.Twelve transgenic lines were obtained (Table 3).

The presence of the transgene in a number of the putative transgeniclines was verified by using the polymerase chain reaction to amplify theinsert from pFL2. The primers used were HF2 =GCTCTTTTGTGCGCTCATTC andHR2=TCGACAGTCACCATTGCTCC (SEQ ID NOS: 45 and 46, respectively, whichwere designed to allow the amplification of a 700 bp fragment.Approximately 100 ng of genomic DNA was added to a solution containing25 pmol of each primer, 1.5 U Taq polymerase (Boehringer Manheim), 200uM of dNTPs, 50 mM KCl, 10 mM Tris.Cl (pH 9), 0.1% (v/v) Triton X-100,1.5 mM MgCl₂, 3% (v/v) formamide, to a final volume of 25 μl.Amplifications conditions were: 4 min denaturation step at 94° C.,followed by 30 cycles of 92° C. for 1 min, 55° C. for 1 min, 72° C. for2 min. A final extension step dosed the program at 72 ° C. for 5 min.All putative transgenic lines tested gave a PCR pattern consistent withthe expected genotype (see Table 3) confirming that the lines were,indeed, transgenic.

                  TABLE 3                                                         ______________________________________                                        Summary of transgenic tobacco lines                                           Line   Construct  Seeds  Seeds                                                obtained                                                                             analyzed   done   produced PCR  Ricinol.                               ______________________________________                                        Wild type                                                                            -          -      +        -    -                                      8      pBI121     +               -                                           2-1    B6         +      +        +    +                                      2-2    B6         +      +        +    +                                      6-1    B6         +               +                                           9-1    A4         +               +                                           9-3    A4         +                                                           10-1   B6         +               +                                           10-2   B6         +               +                                           10-3   B6         +               +                                           10-5   B6         +               +                                           18-1   B6         +               +                                           4/12-1 pBI121     +      +        -    -                                      ______________________________________                                    

Transgenic tissues were analyzed by gas chromatography and massspectrometry for the presence of ricinoleic acid. Calibration standardsfor the gas chromatograph were 1 μl samples of fatty acid methyl estersof an equal mixture of 16:0, 18:0, 18:1, 18:2 and 18:3 (0.125-0.25mg/ml). Ricinoleic acid standards (Sigma) were esterified and silylatedas described below, and injected at 5 or 25 mg/ml. The mass spectrum ofTMS-methyl-ricinoleate is shown in FIG. 18. The fragmentation patternresulting in the ions observed is explained in FIGS. 19A and 19B.

Duplicate seed samples from two independent fah12 transgenic tobaccolines (2-1 and 2-2), one transgenic tobacco line transformed with pBI121(4/12-1) and one wild-type SR-1 tobacco line were used to prepare fattyacid methyl esters (FAMEs). FAMEs were prepared by placing 5 seeds in1.5 ml of 1.0M methanolic HCl in a 13×100 mm glass screw-cap tube cappedwith a teflon-lined cap and heated to 80° C. for 2 hours. Upon cooling,1 ml hexane:isopropanol (3:2) and 0.5 ml 0.2 M Na₂ SO₄ were added andthe FAMEs removed from the hexane phase. Approximately 1 μl of N,O-bis(Trimethylsilyl)trifluoroacetamide (BSTFA) was added (BSTFA,Pierce; 100 ml), to derivatize any hydroxyl groups. The reaction wascarried out at 70 ° C. for 15 min. The products were dried undernitrogen, redissolved in 200 ml hexane and transferred to a gaschromatograph vial. Two ml of each sample were analyzed on a SP2330glass capillary column (30 m, 0.75 mm ID, 0.20 mm film, Supelco), usinga Hewlett-Packard 5890 II series Gas Chromatograph. The samples were notsplit, the temperature program was 150 ° C. (6 min) to 215 ° C. (4°C./min), and flame ionization detectors were used. Care was taken toelute out any carry-over material by injecting three hexane blanks afterthe standards.

                  TABLE 4                                                         ______________________________________                                        Fatty acid composition of fah12 transgenic tobacco seeds                      compared to control seeds. Values are mol % of total fatty acids.                                    fatty                                                                              acids                                             Sample 16:0   18:0     18:1 18:2  18:3 ricinoleate                            ______________________________________                                        WTa    10.1   2.65     12.25                                                                              73.65 1.05 0                                      WTb    10.3   2.75     12.60                                                                              72.79 1.13 0                                      4/12-1a                                                                              10.41  2.40     11.63                                                                              74.23 1.14 0                                      4/12-1b                                                                              10.44  2.70     11.55                                                                              73.57 1.27 0                                      2-1a   10.62  2.83     11.78                                                                              73.19 1.14 0.05                                   2-1b   10.53  2.61     11.81                                                                              73.58 1.09 0.04                                   2-2a   10.95  2.42     10.98                                                                              74.11 1.07 0.09                                   2-2b   11.09  2.92     11.08                                                                              73.56 0.99 0.07                                   ______________________________________                                    

As shown in table 4, seed fatty acid composition for 2-1 and 2-2 aresimilar to both wild-type and 4/21-1 controls. However, a significantdifference was observed in the gas chromatograms in the region of 12.35to 12.44 min. A peak representing about 0.1% of the fatty acid contentin the seed is consistently present in both 2-1 and 2-2 and absent incontrol wild-type and 4/21-1 seeds (FIGS. 20A, 20B and 20C). Undersimilar conditions, the elution time of the TMS-methyl-ricinoleatestandard was 12.43 min. This preliminary result provided the firstindication that fah12-containing transgenic plants 2-1 and 2-2 producedricinoleic acid.

In order to confirm that the observed peak did correspond toTMS-methyl-ricinoleate, mass spectrometry was used. The objective was todetermine if mass spectrums of the compounds eluting in the region of12.35 to 12.44 min in the previous experiment could be unequivocallyattributed to TMS-methyl-ricinoleate. In order to obtain clearerresults, attention was focused on the 2-2 transgenic line seeds, whichcontained about twice as much of the target compound than 2-1 seeds.Seven samples of 20 seeds from the fah12 tobacco transgenic line 2--2were analyzed by gas chromatography and mass spectrometry. Five samplesof 20 wild-type tobacco seeds were used as a control. Three of the seven2--2 samples were ground in 1 ml chloroform:methanol (2:1); the solventwas then evaporated under a stream of nitrogen prior totransesterification with methanolic HCl. The other samples wereextracted directly from intact seeds without grinding as describedabove. Esterification and silylation steps were carried out as describedabove for all samples. To determine how complete TMS-derivatization was,BSTFA was not added for two 2--2 samples (and one ricinoleic acidstandard). Samples were injected into a SP2330 fused silica capillarycolumn (30 m, 0.25 mm ID, 0.20 mm film, Supelco). The temperatureprogram was 100° C. to 150° C. (20° C./min), 150° C. for 6 min, up to190° C. (4 ° C./min), down to 100° C. (20 ° C./min). A Hewlett-Packard5971 series mass selective detector was used in place of the flameionization detector used in the previous experiment. Three hexane blankswere injected between the standard and the wild-type control, and beforethe 2--2 samples.

Grinding the seeds in chloroform:methanol did not result in anysignificant increase in fatty acid concentrations in the samples. Asshown in FIG. 21, TMS-methyl-ricinoleate eluted after 14.602 minutes inthis experiment. In five out of seven cases, TMS-methyl-ricinoleate wasdetected in the 2--2 samples. In two cases the TMS-methyl ricinoleatepeak was obscured by a peak of other material that eluted nearby. TheTMS-methyl ricinoleate peak was never detected in wild-type samples. Oneexample of the total ion chromatogram for one of the 2--2 sample isshown in FIG. 22. At time 14.651 minutes, a signal was detected whichcorresponded to the ion spectrum shown on FIG. 23. The standardTMS-methyl-ricinoleate profile in FIG. 24 is given for a comparison.Three characteristic peaks at M/Z 187, 270 and 299 were consistentlypresent in the mass spectrums when ricinoleic acid was detected. Aconfounding unknown compound elutes after 14.63 minutes in bothwild-type and transgenic seeds. However, its mass spectrum isunequivocally different from TMS-methyl-ricinoleate; in particular, theM/Z=270 ion is totally absent (not shown). The presence of this compoundas well as inefficient TMS-derivatization precluded detection ofTMS-methyl-ricinoleate in two of the 2--2 samples.

These results unequivocally demonstrate the identity of the fah12 cDNAas encoding an oleate hydroxylase. These results also demonstrate thatthe hydroxylase can be functionally expressed in a heterologous plantspecies in such a way that the enzyme is catalytically functional. Theseresults also demonstrate that expression of this hydroxylase gene leadsto accumulation of ricinoleate in a plant species that does not normallyaccumulate hydroxylated fatty acids in extractable lipids.

Although the amount of ricinoleate produced in this example is less thatdesired for commercial production of ricinoleate and other hydroxylatedfatty acids from plants, modifications may be made that will increasethe level of accumulation of hydroxylated fatty acids in plants thatexpress the fah12 or related hydroxylase genes. Improvements in thelevel and tissue specificity of expression of the hydroxylase gene arecontemplated. Methods to accomplish this by the use of strong,seed-specific promoters such as the B. napus napin promoter wili beevident to one skilled in the art. Additional improvements resultingfrom increases in the amount of substrate are also envisioned. Thesubstrate for the hydroxylase is currently believed to be oleate orother monounsaturated fatty acid esterified to phosphatidylcholine.Therefore, expression of the hydroxylase gene in plant species orparticular cultivars that contain elevated levels of oleate-containingphospholipids is believed to lead to increased accumulation ofhydroxylated fatty acids. It is also contemplated that the results maybe improved by modification of the enzymes which cleave hydroxylatedfatty acids from phosphatidylcholine, reduction in the activities ofenzymes which degrade hydroxylated fatty acids and replacement ofacyltransferases which transfer hydroxylated fatty acids to the sn-1 andsn-3 positions of glycerolipids. Although genes for these enzymes arenot currently available, their utility in improving the level ofproduction of hydroxylated fatty adds will be evident based on theresults of biochemical investigations of ricinoleate synthesis.

EXAMPLE 2 Production of Ricinoleate in Arabidopsis thaliana

In order to verify that the fah12 gene can be functionally expressed inother plant species than tobacco, and to demonstrate that increases inthe amount of oleate can affect levels of accumulation of ricinoleate,both wild type and the fad2 mutant of Arabidopsis thaliana (L.) weretransformed with the pFL2 plasmid containing the oleate hydroxylase cDNAinsert. This plasmid was previously used to transform Nicotiana tabacumand is described above.

Inoculums of Agrobacterium tumefaciens strain GV3101, previouslytransformed with pFL2 (see transformation procedure above) were platedon kanamycin LB plates and incubated for 2 days at 30° C. Singlecolonies were used to inoculate large liquid cultures (LB medium with 50mg/l rifampicin, 110 mg/l gentamycin and 200 mg/l kanamycin) to be usedfor the transformation of Arabidopsis plants.

Arabidopsis plants were transformed by the in planta transformationprocedure essentially as described by Bechtold et al., (1993). Cells ofA. tumefaciens GV3101(pFL2) were harvested from liquid cultures bycentrifugation, then resuspended in infiltration medium at OD₆₀₀ =0.8(Infiltration medium was Murashige and Skoog macro and micronutrientmedium containing 10 mg/l 6-benzylaminopurine and 5% glucose). Batchesof 12-15 plants were grown for 3 to 4 weeks in natural light at a meandaily temperature of approximately 25° C. in 3.5 inch pots containingsoil. The intact plants were immersed in the bacterial suspension thentransferred to a vacuum chamber and placed under vacuum produced by alaboratory vacuum pump until tissues appeared uniformly water-soaked(approximately 10 min). The plants were grown at 25° C. under continuouslight (100 μmol m⁻² s⁻¹ irradiation in the 400 to 700 nm range) for fourweeks. The seeds obtained from all the plants in a pot were harvested asone batch. The seeds were sterilized by sequential treatment for 2 minwith ethanol followed by 10 min in a mixture of Bleach, water andTween-80 (50%, 50%, 0.05%) then rinsed thoroughly with sterile water.The seeds were plated at high density (2000 to 4000 per plate) alongwith appropriate control seeds from a known transformed line and a wildtype plant, onto agar-solidified medium in 100 mm petri platescontaining 1/2 X Murashige and Skoog salts medium enriched with B5vitamins and containing kanamycin at 50 mg/l. After a vernalizationperiod of two nights at 4° C., seedlings were grown for a period ofseven days until transformants were clearly identifiable as healthygreen seedlings against a background of chlorotic kanamycin-sensitiveseedlings. The transformants were transferred to soil for two weeksbefore leaf tissue could be used for DNA and lipid analysis.

DNA may be extracted from young leaves from transformants to verify thepresence of an intact fah12 gene. Amplification of the fah12 insert ofthe pFL2 plasmid may be carried out as described above for tobaccotransformants, using the same DNA primers. DNA samples from Arabidopsislines transformed with the unmodified pBI121 vector and from wild typeplants are used as controls, along with appropriate dilutions of pFL2plasmid DNA preparations. The transformants can be positively identifiedafter visualization of a characteristic 1 kb amplified fragment on anethidium bromide stained agarose gel.

Leaves and seeds from fah12 transgenic Arabidopsis plants aresubsequently analyzed for the presence of ricinoleic acid, using gaschromatography. The same procedure, previously described for tobaccoseeds, is used. Fatty add methyl esters are extracted from 100-200 mgleaf tissue or 10-20 seeds, and any hydroxyl groups derivatized using N,O-bis(Trimethylsilyl)trifluoroacetamide (BSTFA, Pierce). Controlsilylated FAMEs from wild type and known pBI121 transgenic lines(transformed with unmodified vector) are analyzed along with both typesof fah12 transgenic lines, with wild type or fad2 backgrounds. ATMS-methyl-ricinoleate standard is used to determine if novel peaks aredue to the accumulation of ricinoleate in the transgenic plant tissue.An equal mass mixture of FAMEs (16:0, 18:0, 18:1, 18:2, 18:3, Sigma) isalso injected to identify any modification in fatty acid composition dueto the expression of fah12.

The average fatty acid composition of leaves in Arabidopsis wild typeand fad2 mutant lines was reported by Miquel and Browse (1992). Fattyacid composition of the different seed lipid fractions was reported byKunst et al., (1992). In contrast with tobacco seeds, 20:1 and 22:1fatty acids accumulate in Arabidopsis seeds. Due to the presence inthese fatty acids of a double bond on carbon 9, it is believed that theyconstitute a new substrate for the oleate-12-hydroxylase encoded byfah12. Studies on the possible substrates for the castor hydroxylasehave shown that mono-unsaturated fatty acids of diverse chain lengthscan be hydroxylated, and that the hydroxyl group is always placed threecarbons distal to the double bond (Howling et al., 1972). Althougholeate was shown be preferred as a substrate, 20:1 and 22:1 should behydroxylated in transgenic Arabidopsis seeds, as they only differ fromoleate by the number of carbon atoms between the double bond and theirmethyl end. However, it is believed that the amount of hydroxylation ofthese fatty acids should be relatively low because they are not normallyesterified to the sn-2 position of phospholipids in Arabidopsis, thepreferred substrate for the castor hydroxylase. This limitation can beovercome by introducing a gene for an sn-2 acyltransferase that does notexclude 20:1 and 22:1 from the sn-2 position of glycerolipids.

The presence of ricinoleate in the leaves or in the seeds may beverified in all cases by mass spectrometry, using the method describedabove (see Example 1). Similarly, any late-eluting compound found inchromatograms from transgenic lines but not in the controls may also besubjected to mass-spectrometry, and mass-spectra analyzed for thepresence of ions characteristic of TMS-derivatized hydroxylated fattyacids. It is contemplated that higher levels of oleate in fad2 mutantsincrease the level of accumulation of ricinoleic acid.

This example illustrates, in a different plant system, the expression ofthe fah12 gene encoding an active castor oleate-12-hydroxylase. AlthoughArabidopsis is not an economically important plant species, it is widelyaccepted by plant biologists as a model for higher plants. Therefore,this example demonstrates the general utility of the invention describedhere to the modification of oil composition in higher plants. Oneadvantage of studying the expression of this novel gene in Arabidopsisis the existence in this system of a large body of knowledge on lipidmetabolism, as well as the availability of a collection of mutants whichcan be used to provide useful information on the biochemistry of fattyacid hydroxylation in plant species. Another advantage is the ease oftransposing any of the information obtained on metabolism of ricinoleatein Arabidopsis to Brasslea species such as the crop plant Brassica napusin order to mass produce ricinoleate for industrial use.

EXAMPLE 3 Obtaining Other Plant Fatty Acyl Hydroxylases

Having obtained sequence (amino acid and DNA) for castor oleatehydroxylase, fatty acyl hydroxylase genes from other plant sources canbe readily isolated. In this example, three methods are described toisolate other hydroxylase genes: (A) by DNA hybridization techniquesusing sequences or peptide sequence information from the castorhydroxylase gene, (B) by polymerase chain reaction based on sequencesimilarities between the castor oleate hydroxylase gene and theArabidopsis Δ12 desaturase, and (C) by immunological cross-reactivityusing antibodies to the castor protein as a probe.

In any of these methods, cDNA or genomic libraries from the desiredplants are generally necessary. Many methods of constructing cDNA orgenomic libraries are provided in the scientific literature (for examplesee Huyuh et at., 1985) and many kits for synthesis of cDNA librariesare available commercially (eg. In Vitrogen, Pharmacia, Stratagene).

Isolation of Hydroxylase Genes by Heterologous Hybridization

The full-length cDNA done for the castor hydroxylase is a preferredheterologous hybridization probe. However, fragments of the cDNA arealso useful as heterologous hybridization probes. In order to determineif the castor cDNA is a suitable probe for a given species, Northernanalysis of RNA from various tissues of the target plant species isconducted to determine appropriate hybridization conditions. Sincehydroxylated fatty adds generally accumulate preferentially in seeds butnot in leaves, RNA is isolated from developing embryo tissues and leavesas described in Example 1, electrophoresed in a formaldehyde/agarose geland transferred to a nylon membrane filter as described in Example 1.The ³² P-labeled oleate hydroxylase probe (Sambrook et al., 1989) isadded to a hybridization solution containing 50% formamide, 6×SSC (or6×SSPE), 5×Denhardt's reagent, 0.5% SDS, and 100 5μg/ml denatured salmonsperm DNA fragments. The hybridization solution containing the labeledprobe is incubated with the Northern filter at approximately 40° C. for18 hours or longer to allow hybridization of the probe to sequenceswhich show regions of significant homology (more than about 60%identity). The filter is then washed at low stringency (room temperatureto 42 ° C. in 1X SSC). After exposing the filter to an X-ray film forvarious amounts of time, stringency conditions can be adjusted bydecreasing the amount of formamide progressively to zero and duplicatefilters probed until a limited number of distinct bands can bereproducibly detected. The presence of a higher degree of hybridizationto the lane of RNA from tissues that accumulate hydroxylated fatty acidsis taken as preliminary evidence that the probe is detecting transcriptsfrom a hydroxylase gene.

If one or several mRNA species do hybridize to the DNA probe under thechosen stringency conditions, a cDNA library (or genomic library) isthen constructed from the target plant tissue using purified poly-A RNA(hisant and Houdebine, 1990) that was isolated at the stage whenhydroxylation of fatty acids is known to occur. The cDNA library isscreened using labelled fah12 cDNA as a probe under conditionsestablished for Northern blots. As mentioned above, a number of methodsexist for labelling the DNA fragment, utilizing for example ³² P ordigoxigenin-labelled deoxynucleotides. Typically, 50,000 to 100,000plaques are plated on an E. coli host strain (eg. strain XL1-blue wouldbe a suitable host if the Stratagene λZapII vector is used forconstructing the library). After transfer of the plaques onto Nylonmembranes, hybridization to the probe is carried out in the appropriatehybridization buffer, for example 4×SET, 0.2% SDS, 0.1% sodiumpyrophosphate and 100 mg/ml heparin (see Southern hybridization sectionabove), for a period of 16 to 24 hours. Again, care should be taken tochoose low stringency conditions including the appropriate hybridizationtemperature, such as 55° C., and subsequent washing conditions (roomtemperature to 55° C. in 1-2×SSC). Several adjustments may have to bemade until only a small number of phage clones are detected which can beanalyzed further. Sequence information should be collected at this pointon isolated clones (see Example 1) to verify that they encode a relatedfatty acid hydroxylase. In this case a full-length cDNA should beisolated from the library for the production of transgenic plants whichcan in turn be analyzed for any accumulation of hydroxylated fatty acids(see examples 1 and 2). Similar procedures are followed for theproduction and screening of a genomic library. The genomic clone canalso be used for the production of hydroxylated fatty acids intransgenic plants when expressed either under its own promoter or underthe control of another promoter such as the B. napus napin promoter, theArabidopsis 12S promoter, the soybean 7S storage protein promoter or anyof the many other promoters which have been characterized.

It is contemplated that, genes encoding Δ12-desaturase may also bedetected and recovered due to sequence hornology between the Δ12desaturase and 12-hydroxylase genes. Hydroxylase and desaturase genescan be distinguished by cloning and sequencing the corresponding cDNAclones and comparing them to the known hydroxylase and desaturase genes.Hydroxylase genes can be recognized by having a higher degree of overallsequence identity to the castor oleate hydroxylase gene than to theArabidopsis Δ12 desaturase gene.

Isolation of Hybridization Probes by PCR Methods

An alternative approach to heterologous hybridization is to amplify thetarget gene using degenerate PCR primers. Based on the high degree ofamino acid sequence identity between the Arabidopsis fad2 gene and thecastor oleate hydroxylase, probes for oleate hydroxylases can beobtained by preparing mixed oligonucleotides of greater than 10,preferably of 15 or more, nucleotides in length representing allpossible nucleotide sequences which could encode the corresponding aminoacid sequences. This method is clearly documented by Gould et al.(1989). Typically, mixed oligonucleotide primers of 15 to 40 nucleotidesare used in PCR reactions. For example the following oligonucleotidepairs (or fragments thereof) are contemplated as generally useful toamplify a 0.65 kb fragment of cDNAs for acyl hydroxylases from otherplants. These primers may also be used to amplify a ≧0.65 kb genomicfragment from other species (the exact size of a genomic clone cannot bedetermined beforehand because the size and position of introns in thegenome of these species is not known).

PRIMER- 1:

5'-TGGAA(GA)TA(CT)(TA)(GC)(AGCT)CA(CT)(AC)G(AGCT)(AC) G(ACGT)CA(AC)CA-3'

PRIMER-2:

5'AA(GACT)A(AG)(AG)TG(AG)TG(ACGT)GC(ACGT)AC(AG)TG(ACGT) GT(AG)TC-3' (SEQID NOS: 47 and 48)

These examples are intended only to illustrate the method and are notintended to be an exhaustive list of all possible oligonucleotideprimers that would be suitable for this purpose. Typically, one skilledin the art would prepare a number of such primers based on the regionsof conserved sequence between the castor fah12 and Arabidopsis (orother) Δ12 desaturase gene products, and would then test variouscombinations of these primers for their ability to produce a PCR productof the expected size. When a PCR product of the expected size wasproduced, the band would be excised from an agarose gel, cloned and thenucleotide sequence determined. As noted above, comparison of thesequence of the fragment permits identification of the fragment as beingpart of a hydroxylase gene or a desaturase gene. The cloned fragment maythen be used as a hybridization probe under conditions of highstringency (ie., 68° C. in 5×SCC) to isolate cDNA or genomic clones fromthe target species. As noted above, these clones may be identified ashydroxylase or desaturase dones by sequence similarity to knownhydroxylase and desaturase genes. The identity of a particular done isthen verified by expression of the done in a suitable transgenic host asdescribed in Example 1. The choice of a suitable host for expression ofthe gene is mediated by the availability in the host of the substratefor the hydroxylase enzyme and the ability to transform the particularhost. In view of recent progress in transforming many plant species,methods of transformation are not thought to be a limitation.

Use of Immunological Methods to Identify Hydroxylase Genes

Acyl hydroxylase genes can also be identified by immunologicalcross-reactivity using antibodies to the enzyme as a probe. Thisexperiments involves three steps: (1) isolation of large quantities ofthe castor protein from recombinant E. coli strains for the fah12 geneor from castor; (2) production of antibodies against the protein byinoculated rabbits; (3) using the labelled antibodies as a probe on anexpression library of mRNA sequences from the target plant. Because ofthe relative ease of production of large quantities of protein from acloned gene, the use of recombinant protein is the preferred method.

In the first step, the fah12 insert of the pFL2 clone can be transferredby appropriate cloning techniques into one of the numerous commerciallyavailable plasmid expression vectors (such as the pET3; Fox et al.,1993), then transformed into the appropriate E. coli strain (Fox et al.,1993). Sequences on the vector, such as appropriatetranscription/translation termination sequences downstream of the insertand promoter sequences upstream (such as the lac promoter) should bepresent to allow regulated accumulation of recombinant protein.

After growing large liquid cultures of the recombinant strain, a varietyof protein purification techniques can be used. Typically, proteinsaccumulated in E. coli inclusion bodies are released and collected afterlysis of the cells by a centrifugation step. In the second step, rabbitsare serially injected using native or denatured proteins. Antibodies canbe recovered from the immunized rabbit sera, using for example beadscoated with protein A, a component of the cell wall of S. aureus thatbind strongly to the constant region of the IgG heavy chain.Alternatively, antibodies can be purified by affinity to antigenimmobilized on nitrocellulose filters. The suitability of the purifiedantibody as a probe should then be tested by hybridizing it sequentiallyto denatured castor proteins, in vitro translation products of theoriginal fah12 mRNA and to translated vector sequences.

Before constructing a cDNA expression library from which cDNAs clonesencoding a novel hydroxylase may be isolated, the produced antibodiesshould be probed onto a Western blot carrying bound proteins from thetarget plant. To that effect, an appropriate amount of tissue yieldingabout 100 μg of proteins is ground in liquid nitrogen, then dispersed insuspension buffer (0.1M NaCl, 0.01 M Tris. HCl (pH 7.6), 0.001M EDTA (pH8.0), 1 μg/ml aprotinin, 100 μg/ml PMSF) before being added to an equalvolume of 2×SDS gel-loading buffer (100 mM Tris. Cl (pH 6.8, 200 mM DDT,4% SDS, 0.2% BPB, 20% glycerol). After sonication, the sample is addedto an SDS-acrylamide gel of the appropriate concentration (10%acrylamide for example). After electrophoresis and staining of the gel,the separated proteins can be transferred onto a nitrocellulose filterwhich will be incubated in hybridization buffer containing thehydroxylase-raised antibody.

First, hybridization is carried out in a 1:100 to 1:5000 solution of theantibody in the following buffer: 5% nonfat dried milk, 0.01% antifoamA, 0.02% sodium azide in PBS). The incubation temperature is 4° C. Inorder to increase the sensitivity of the detection, which is importantin the present case of heterologous antibody-antigen hybridization,increasing incubation times would be explored to determine the optimumconditions.

After this primary incubation, the antibody-antigen complexes can bedetected in a variety of ways, using ¹²⁵ I-labelled anti-immunoglobin orprotein A, or more commonly one of these two secondary reagentconjugated to horseradish peroxidase or alkaline phosphatase. In thelatter case, the appropriate substrate to the conjugated enzyme is addedprior to exposure of the filter to X-ray films.

In the case when one or several protein species can be detected on thewestern blot, an expression cDNA library can be constructed withpurified polyA RNA from tissue(s) accumulating the hydroxylated fattyacid. cDNAs should preferentially be cloned in a bacteriophage vectorrather than a plasmid vector, as larger numbers of clones canconveniently be screened. As an example, lambda gt11 and its derivativescan be used (Huyuh et al., 1985). In these vectors expression of clonedcDNA species is under the control of the lac repressor. Again, a typical50,000 to 100,000 clones can be screened for expression of a fatty acylhydroxylase. In the presence of IPTG, recombinant phages express theforeign gene, and the resulting proteins can be imprinted onnitrocellulose filters for subsequent western hybridization, in theconditions described above. As mentioned in the previous sectiondescribing how labelled DNA probes can be used, positive clones shouldthen be analyzed further to determine if they do encode an hydroxylasewith similarity to the castor enzyme.

It is contemplated that the foregoing methods can be used to allow theisolation of acyl hydroxylase genes from species other than castor wherehydroxylated fatty acids can be found. As mentioned earlier, at least 33structurally distinct monohydroxylated plant fatty acids have beendescribed (Gunstone et al., 1986; Smith, 1985; van de Loo et al., 1993).The approaches described above can be of utility to isolate the genesencoding the corresponding hydroxylases. These species would be ofprimary interest for the isolation of genes related to fah12, especiallythe species in the Lesquerella genus. Members of this genus accumulateoil, which like castor oil, contains a hydroxyl group on the fatty acidthree carbons distal to the first double bond from the carboxy end.Lesquerella densipila is of particular interest since it accumulates the16 carbon hydroxy fatty acid equivalent of ricinoleic acid. Similarly,this species also accumulates a di-unsaturated version of ricinoleic loacid. The enzyme involved in the biosynthesis of the isomerisoricinoleic acid in Strophanthus species is also expected to havestructural and catalytic similarity to the castor oleate-12-hydroxylase.In that case, the hydroxyl group and the double bond are inverted withrespect to ricinoleic acid.

Clones identified using DNA hybridization or irnmunological screeningtechniques are then purified, the DNA isolated, and the sequence of thegenes is determined as described in Example 1. In this manner, it isverified that the clones encode a related fatty acyl hydroxylase. Thenewly isolated plant hydroxylase sequences can also be used to isolategenes for fatty acyl hydroxylases from other plant species using thetechniques described above.

The above examples demonstrate critical factors in the production ofhydroxylated fatty acids. A complete cDNA sequence of the castor oleatehydroxylase is also provided with a demonstration of the activity of thepolypeptide encoded thereby in transgenic plants. A full sequence of thecastor hydroxylase is also given with various constructs for use in hostcells. Through this invention, one can obtain the amino acid and nucleicacid sequences which encode plant fatty acyl hydroxylases from a varietyof sources and for a variety of applications. Accordingly, within itsvarious embodiments, it will be appreciated that the invention includessuch features as: recombinant DNA constructs comprising at least aportion of a plant fatty acyl hydroxylase encoding sequence, preferablybut not necessarily a plant oleate hydroxylase encoding sequence;transgenic host cells including such construct and containing anexpressed plant fatty acyl hydroxylase; methods of producing planthydroxylase in such host cells or progeny thereof; methods of increasingthe fatty acid content in plant cells or in triglycerides produced fromplants, e.g. oilseed crop plants, using the present constructs. Oilseedcrop plants which are contemplated include rapeseed, Canola, flax,sunflower, safflower, cotton, cuphea, soybean, peanut, coconut, oil palmand corn. Other features of the invention such as the possibility ofusing constructs according to the invention or nucleotides or deducedamino acid sequences derived therefrom to identify and isolate acylhydroxylase genes from plant species other than Ricinus communis (L),will also be evident from the foregoing.

All publications mentioned in this specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications are herein incorporated by reference to thesame extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

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Thiede, M. A., Ozols, J., Strittmatter, P. (1986) Construction andsequence of cDNA for rat liver stearoyl coenzyme A desaturase. J. Biol.Chem. 261, 13230-13235.

van de Loo, F. J., Fox, B. G., Somerville, C. (1993) Unusual fattyacids, in Lipid Metabolism in Plants, T. S. Moore Jr., Ed., CRC Press,Boca Raton, pp91-126.

van de Loo, F., and Somerville, C. (1994) A plastid omega-3 desaturasefrom castor (Ricinus communis L.). Plant Physiol 105, 443-444.

von Heijne, G. (1985) Signal sequences. J. Mol. Biol. 184,99-105.

Yadav, N. S., Wierzbicki, A., Aegerter, M., Caster, C. S., Perez-Grau,L., Kinney, A. J., Hitz, W. D., Booth, R., Schweiger, B., Stecca, K. L.,Allen, S. M., Blackwell, M., Reiter, R. S., Carlson, T. J., Russell, S.H., Feldmann, K. A., Pierce, J., Browse, J. (1993) Cloning of higherplant ω3 fatty acid desaturases. Plant Physiol. 103, 467-476.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 48                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 523 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGACCTCGGAATCTTTGCCACAACGTTTGTGCTTTATCAGGCTACAATGGCAAAAGGGTT60                GGCTTGGGTAATGCGTATCTATGGGGTGCCATTGCTTATTGTTAACTGTTTCCTTGTTAT120               GATCACATACTTGCAGCACACTCACCCAGCTATTCCACGCTATGGCTCATCGGAATGGGA180               TTGGCTCCGGGGAGCAATGGTGACTGTCGATAGAGATTATGGGGTGTTGAATAAAGTATT240               CCATAACATTGCAGNCACTCATGTAGCTCATCANCTCTTTGCTACAGTGNCACATTACCA300               TGCAATGGGGGNCNCTAAGCAATCAAGGCCTATAATGGGNGGATNTTACCGGATNATNGG360               NCCCCATTTACAAGGGATTTTTGGGGGGCAAANNNAGTCNTTTTNTNCTGGCCAATTAAG420               GGGNCTCAAAAAGGGTTTNTTGGCCCGCAAGTTTAAAAGGNATTTGNCNGTTTTTAGGGN480               GGATTTNCCAAAGGATTTTTTTNGGAATTNTNTTTNAGGGGGG523                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 540 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTGACCTCGGAATCTTTGCCACAACGTTTGTCCTTTATCAGGCTACAATGGCAAAAGGGT60                TGGCTTGGGTAATGCGTATCTATGGGGTGCCATTGCTTATTGTTAACTGTTTCCTTGTTA120               TGATCACATACTTGCAGCACACTCACCCAGCTATTCCACGCTATGGCTCATCGGAATGGG180               ATTGGCTCCGGGGAGCAATGGTGACTGTCGATAGAGATTATGGGGTGTTGAATAAAGTAT240               TCCATAACATTGCAGACACTCATGTAGCTCATCATCTCTTTGCTACAGTGCCACATTACC300               ATGCAATGGAGGCCACTAAAGCAATCAAGCCTATAATGGGTGAGTATTACCGGTATGATG360               GTNCCCATTTTACAAGGCATTGTGGAGGGAGCAAAGGAGTCTTNCCGNCGGCCAANTGAG420               NNGNCNCANAAGNGGTTTTGGCCCGACAAGTTTAAAAGGCATNNCCTGTTTTNAGGGGGA480               TTNCAANAGGATTTTTNNGGAATNGCTTTNGGGGNAAAANCAGCATTGNGTTAAGGNNGC540               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       IleProArgTyrGlySerSerGluTrpAspTrpLeuArgGlyAlaMet                              151015                                                                        ValThrValAspArgAspTyrGlyValLeuAsnLysValPheHisAsn                              202530                                                                        IleAlaXaaThrHisValAlaHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuProTrpTyrArgGlyGlnGluTrpSerTyrLeuArgGlyGlyLeu                              151015                                                                        ThrThrValAspArgAspTyrGlyTrpIleAsnAsnValHisHisAsp                              202530                                                                        IleGlyThrHisValIleHisHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       HisAsnIleAlaXaaThrHisValAlaHisXaaLeuPheAlaThrVal                              151015                                                                        XaaHisTyrHisAlaMetGlyXaaXaaLysGlnSerArgProIleMet                              202530                                                                        GlyGlyXaaTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       HisHisAspIleGlyThrHisValIleHisHisLeuPheProGlnIle                              151015                                                                        ProHisTyrHisLeuValGluAlaThrLysSerAlaLysSerValLeu                              202530                                                                        GlyLysTyrTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ValLeuTyrGlnAlaThrMetAlaLysGlyLeuAlaTrpValMetArg                              151015                                                                        IleTyrGlyValProLeuLeuIleValAsnCysPheLeuValMetIle                              202530                                                                        ThrTyrLeuGlnHis                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ValLeuLeuTyrLeuSerLeuThrIleGlyProIlePheMetLeuLys                              151015                                                                        LeuTyrGlyValProTyrLeuIlePheValMetTrpLeuAspPheVal                              202530                                                                        ThrTyrLeuHisHis                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       IleProArgTyrGlySerSerGluTrpAspTrpLeuArgGlyAlaMet                              151015                                                                        ValThrValAspArgAspTyrGlyValLeuAsnLysValPheHisAsn                              202530                                                                        IleAlaXaaThrHisValAlaHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LeuProTrpTyrArgGlyLysGluTrpSerTyrLeuArgGlyGlyLeu                              151015                                                                        ThrThrIleAspArgAspTyrGlyIlePheAsnAsnIleHisHisAsp                              202530                                                                        IleGlyThrHisValIleHisHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      HisAsnIleAlaXaaThrHisValAlaHisXaaLeuPheAlaThrVal                              151015                                                                        XaaHisTyrHisAlaMetGlyXaaXaaLysGlnSerArgProIleMet                              202530                                                                        GlyGlyXaaTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      HisHisAspIleGlyThrHisValIleHisHisLeuPheProGlnIle                              151015                                                                        ProHisTyrHisLeuValAspAlaThrArgAlaAlaLysHisValLeu                              202530                                                                        GlyArgTyrTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ValMetArgIleTyrGlyValProLeuLeuIleValAsnCysPheLeu                              151015                                                                        ValMetIleThrTyrLeuGlnHis                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ValLeuLysValTyrGlyValProTyrIleIlePheValMetTrpLeu                              151015                                                                        AspAlaValThrTyrLeuHisHis                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      HisAsnIleAlaAspThrHisValAlaHisHisLeuPheAlaThrVal                              151015                                                                        ProHisTyrHisAlaMetGluAlaThrLysAlaIleLysProIleMet                              202530                                                                        GlyGluTyrTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      HisHisAspIleGlyThrHisValIleHisHisLeuPheProGlnIle                              151015                                                                        ProHisTyrHisLeuValAspAlaThrArgAlaAlaLysHisValLeu                              202530                                                                        GlyArgTyrTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      IleProArgTyrGlySerSerGluTrpAspTrpLeuArgGlyAlaMet                              151015                                                                        ValThrValAspArgAspTyrGlyValLeuAsnLysValPheHisAsn                              202530                                                                        IleAlaAspThrHisValAlaHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      LeuProTrpTyrArgGlyLysGluTrpSerTyrLeuArgGlyGlyLeu                              151015                                                                        ThrThrIleAspArgAspTyrGlyIlePheAsnAsnIleHisHisAsp                              202530                                                                        IleGlyThrHisValIleHisHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ValMetArgIleTyrGlyValProLeuLeuIleValAsnCysPheLeu                              151015                                                                        ValMetIleThrTyrLeuGlnHis                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ValLeuLysValTyrGlyValProTyrIleIlePheValMetTrpLeu                              151015                                                                        AspAlaValThrTyrLeuHisHis                                                      20                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      HisAsnIleAlaAspThrHisValAlaHisHisLeuPheAlaThrVal                              151015                                                                        ProHisTyrHisAlaMetGluAlaThrLysAlaIleLysProIleMet                              202530                                                                        GlyGluTyrTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      HisHisAspIleGlyThrHisValIleHisHisLeuPheProGlnIle                              151015                                                                        ProHisTyrHisLeuValGluAlaThrLysSerAlaLysSerValLeu                              202530                                                                        GlyLysTyrTyrArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      IleProArgTyrGlySerSerGluTrpAspTrpLeuArgGlyAlaMet                              151015                                                                        ValThrValAspArgAspTyrGlyValLeuAsnLysValPheHisAsn                              202530                                                                        IleAlaAspThrHisValAlaHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuProTrpTyrArgGlyGlnGluTrpSerTyrLeuArgGlyGlyLeu                              151015                                                                        ThrThrValAspArgAspTyrGlyTrpIleAsnAsnValHisHisAsp                              202530                                                                        IleGlyThrHisValIleHisHis                                                      3540                                                                          (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      ValLeuTyrGlnAlaThrMetAlaLysGlyLeuAlaTrpValMetArg                              151015                                                                        IleTyrGlyValProLeuLeuIleValAsnCysPheLeuValMetIle                              202530                                                                        ThrTyrLeuGlnHis                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ValLeuLeuTyrLeuSerLeuThrIleGlyProIlePheMetLeuLys                              151015                                                                        LeuTyrGlyValProTyrLeuIlePheValMetTrpLeuAspPheVal                              202530                                                                        ThrTyrLeuHisHis                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 523 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      TGACCTCGGAATCTTTGCCACAACGTTTGTGCTTTATCAGGCTACAATGGCAAAAGGGTT60                GGCTTGGGTAATGCGTATCTATGGGGTGCCATTGCTTATTGTTAACTGTTTCCTTGTTAT120               GATCACATACTTGCAGCACACTCACCCAGCTATTCCACGCTATGGCTCATCGGAATGGGA180               TTGGCTCCGGGGAGCAATGGTGACTGTCGATAGAGATTATGGGGTGTTGAATAAAGTATT240               CCATAACATTGCAGNCACTCATGTAGCTCATCANCTCTTTGCTACAGTGNCACATTACCA300               TGCAATGGGGGNCNCTAAGCAATCAAGGCCTATAATGGGNGGATNTTACCGGATNATNGG360               NCCCCATTTACAAGGGATTTTTGGGGGGCAAANNNAGTCNTTTTNTNCTGGCCAATTAAG420               GGGNCTCAAAAAGGGTTTNTTGGCCCGCAAGTTTAAAAGGNATTTGNCNGTTTTTAGGGN480               GGATTTNCCAAAGGATTTTTTTNGGAATTNTNTTTNAGGGGGG523                                (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 517 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TGACCTCGGAATCTTTGCCACAACGTTTGTCCTTTATCAGGCTACAATGGCAAAAGGGTT60                GGCTTGGGTAATGCGTATCTATGGGGTGCCATTGCTTATTGTTAACTGTTTCCTTGTTAT120               GATCACATACTTGCAGCACACTCACCCAGCTATTCCACGCTATGGCTCATCGGAATGGGA180               TTGGCTCCGGGGAGCAATGGTGACTGTCGATAGAGATTATGGGGTGTTGAATAAAGTATT240               CCATAACATTGCAGACACTCATGTAGCTCATCATCTCTTTGCTACAGTGCCACATTACCA300               TGCAATGGAGGCCACTAAAGCAATCAAGCCTATAATGGGTGAGTATTACCGGTATGATGG360               TNCCCATTTTACAAGGCATTGTGGAGGGAGCAAAGGAGTCTTNCCGNCGGCCAANTGAGN420               NGNCNCANAAGNGGTTTTGGCCCGACAAGTTTAAAAGGCATNNCCTGTTTTNAGGGGGAT480               TNCAANAGGATTTTTNNGGAATNGCTTTNGGGGNAAA517                                      (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      ACTTGGTGATGATAGTTCCGGTTATAGCAAATCCGACCAAAAACGGCCAGTTACGGTTGA60                ACTCCCGCTTGAAGAACACGGGCCATGGATCGAACCACCTTTTCATCTTTTCTCGAAGCC120               TCAGGAAAGTGTTTAAAAAAGAGCTTTAGA150                                             (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CACACTTGGTGACCTCAAATCAAACACCACACCTTATAACTTAGTCTTAAGAGAGAGAGA60                GAGAGAGAGGAGACATTTCTCTTCTCTGAGATAAGCACTTCTCTTCCAGACATCGAAGCC120               TCAGGAAAGTGCTTAAAAAGAGCTTAAGAA150                                             (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 104 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TTAAGAGAGAGAGAGAGAGAGAGGAGACATTTCTCTTCTCTGAGATAAGCACTTCTCTTC60                CAGACATCGAAGCCTCAGGAAAGTGCTTAAAAAGAGCTTAAGAA104                               (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TTCTCTTCCAGACATCGAAGCCTCAGGAAAGTGCTTAAAAAGAGCTTAAGAA52                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CTCTAAAGGCACTTCTCTTCCAGACATCGAAGCCTCAGGAAAGTGCTTAAAAAGAGCTTA60                AGAA64                                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GAGAGAGAGGAGACATTTCTCTTCTCTGAGATAAGCACTTCTCTTCCAGACATCGAAGCC60                TCAGGAAAGTGCTTAAAAAGAGCTTAAGAA90                                              (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 83 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AGGAGACACTTCTCTTCTCTGAGATAAGCACTTCTCTTCCAGACATCGAAGCCTCAGGAA60                AGTGCTTAAAAAGAGCTTAAGAA83                                                     (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CAGACATCGAAGCCTCAGGAAAGTGCTTAAAAAGAGCTTAAGAA44                                (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      ACTTCTCTTCCAGACATCGAAGCCTCAGGAAAGTGCTTAAAAAGAGCTTAAGAA54                      (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 79 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      GACATTTCTCTTCTCTGAGATAAGCACTTCTCTTCCAGACATCGAAGCCTCAGGAAAGTG60                CTTAAAAAGAGCTTAAGAA79                                                         (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1448 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 187..1350                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GCCACCTTAAGCGAGCGCCGCACACGAAGCCTCCTTTCACACTTGGTGACCTCAAATCAA60                ACACCACACCTTATAACTTAGTCTTAAGAGAGAGAGAGAGAGAGAGGAGACATTTCTCTT120               CTCTGAGATAAGCACTTCTCTTCCAGACATCGAAGCCTCAGGAAAGTGCTTAAAAAGAGC180               TTAAGAATGGGAGGTGGTGGTCGCATGTCTACTGTCATAACCAGCAAC228                           MetGlyGlyGlyGlyArgMetSerThrValIleThrSerAsn                                    1510                                                                          AACAGTGAGAAGAAAGGAGGAAGCAGCCACCTTAAGCGAGCGCCGCAC276                           AsnSerGluLysLysGlyGlySerSerHisLeuLysArgAlaProHis                              15202530                                                                      ACGAAGCCTCCTTTCACACTTGGTGACCTCAAGAGAGCCATCCCACCC324                           ThrLysProProPheThrLeuGlyAspLeuLysArgAlaIleProPro                              354045                                                                        CATTGCTTTGAACGCTCTTTTGTGCGCTCATTCTCCTATGTTGCCTAT372                           HisCysPheGluArgSerPheValArgSerPheSerTyrValAlaTyr                              505560                                                                        GATGTCTGCTTAAGTTTTCTTTTCTACTCGATCGCCACCAACTTCTTC420                           AspValCysLeuSerPheLeuPheTyrSerIleAlaThrAsnPhePhe                              657075                                                                        CCTTACATCTCTTCTCCGCTCTCGTATGTCGCTTGGCTGGTTTACTGG468                           ProTyrIleSerSerProLeuSerTyrValAlaTrpLeuValTyrTrp                              808590                                                                        CTCTTCCAAGGCTGCATTCTCACTGGTCTTTGGGTCATCGGCCATGAA516                           LeuPheGlnGlyCysIleLeuThrGlyLeuTrpValIleGlyHisGlu                              95100105110                                                                   TGTGGCCATCATGCTTTTAGTGAGTATCAGCTGGCTGATGACATTGTT564                           CysGlyHisHisAlaPheSerGluTyrGlnLeuAlaAspAspIleVal                              115120125                                                                     GGCCTAATTGTCCATTCTGCACTTCTGGTTCCATATTTTTCATGGAAA612                           GlyLeuIleValHisSerAlaLeuLeuValProTyrPheSerTrpLys                              130135140                                                                     TATAGCCATCGCCGCCACCATTCTAACATAGGATCTCTCGAGCGAGAC660                           TyrSerHisArgArgHisHisSerAsnIleGlySerLeuGluArgAsp                              145150155                                                                     GAAGTGTTCGTCCCGAAATCAAAGTCGAAAATTTCATGGTATTCTAAG708                           GluValPheValProLysSerLysSerLysIleSerTrpTyrSerLys                              160165170                                                                     TACTCAAACAACCCGCCAGGTCGAGTTTTGACACTTGCTGCCACGCTC756                           TyrSerAsnAsnProProGlyArgValLeuThrLeuAlaAlaThrLeu                              175180185190                                                                  CTCCTTGGCTGGCCTTTATACTTAGCTTTCAATGTCTCTGGTAGACCT804                           LeuLeuGlyTrpProLeuTyrLeuAlaPheAsnValSerGlyArgPro                              195200205                                                                     TACGATCGCTTTGCTTGCCATTATGATCCCTATGGCCCAATATTTTCC852                           TyrAspArgPheAlaCysHisTyrAspProTyrGlyProIlePheSer                              210215220                                                                     GAAAGAGAAAGGCTTCAGATTTACATTGCTGACCTCGGAATCTTTGCC900                           GluArgGluArgLeuGlnIleTyrIleAlaAspLeuGlyIlePheAla                              225230235                                                                     ACAACGTTTGTGCTTTATCAGGCTACAATGGCAAAAGGGTTGGCTTGG948                           ThrThrPheValLeuTyrGlnAlaThrMetAlaLysGlyLeuAlaTrp                              240245250                                                                     GTAATGCGTATCTATGGGGTGCCATTGCTTATTGTTAACTGTTTCCTT996                           ValMetArgIleTyrGlyValProLeuLeuIleValAsnCysPheLeu                              255260265270                                                                  GTTATGATCACATACTTGCAGCACACTCACCCAGCTATTCCACGCTAT1044                          ValMetIleThrTyrLeuGlnHisThrHisProAlaIleProArgTyr                              275280285                                                                     GGCTCATCGGAATGGGATTGGCTCCGGGGAGCAATGGTGACTGTCGAT1092                          GlySerSerGluTrpAspTrpLeuArgGlyAlaMetValThrValAsp                              290295300                                                                     AGAGATTATGGGGTGTTGAATAAAGTATTCCATAACATTGCAGACACT1140                          ArgAspTyrGlyValLeuAsnLysValPheHisAsnIleAlaAspThr                              305310315                                                                     CATGTAGCTCATCATCTCTTTGCTACAGTGCCACATTACCATGCAATG1188                          HisValAlaHisHisLeuPheAlaThrValProHisTyrHisAlaMet                              320325330                                                                     GAGGCCACTAAAGCAATCAAGCCTATAATGGGTGAGTATTACCGGTAT1236                          GluAlaThrLysAlaIleLysProIleMetGlyGluTyrTyrArgTyr                              335340345350                                                                  GATGGTACCCCATTTTACAAGGCATTGTGGAGGGAGGCAAAGGAGTGC1284                          AspGlyThrProPheTyrLysAlaLeuTrpArgGluAlaLysGluCys                              355360365                                                                     TTGTTCGTCGAGCCAGATGAAGGAGCTCCTACACAAGGCGTTTTCTGG1332                          LeuPheValGluProAspGluGlyAlaProThrGlnGlyValPheTrp                              370375380                                                                     TACCGGAACAAGTATTAAAAAAGTGTCATGTAGCCTGTTTCTTTAAGAGAAGTAA1387                   TyrArgAsnLysTyr                                                               385                                                                           TTAGAACAAGAAGGAATGTGTGTGTAGTGTAATGTGTTCTAATAAAGAAGGCAAAAAAAA1447              A1448                                                                         (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 387 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      MetGlyGlyGlyGlyArgMetSerThrValIleThrSerAsnAsnSer                              151015                                                                        GluLysLysGlyGlySerSerHisLeuLysArgAlaProHisThrLys                              202530                                                                        ProProPheThrLeuGlyAspLeuLysArgAlaIleProProHisCys                              354045                                                                        PheGluArgSerPheValArgSerPheSerTyrValAlaTyrAspVal                              505560                                                                        CysLeuSerPheLeuPheTyrSerIleAlaThrAsnPhePheProTyr                              65707580                                                                      IleSerSerProLeuSerTyrValAlaTrpLeuValTyrTrpLeuPhe                              859095                                                                        GlnGlyCysIleLeuThrGlyLeuTrpValIleGlyHisGluCysGly                              100105110                                                                     HisHisAlaPheSerGluTyrGlnLeuAlaAspAspIleValGlyLeu                              115120125                                                                     IleValHisSerAlaLeuLeuValProTyrPheSerTrpLysTyrSer                              130135140                                                                     HisArgArgHisHisSerAsnIleGlySerLeuGluArgAspGluVal                              145150155160                                                                  PheValProLysSerLysSerLysIleSerTrpTyrSerLysTyrSer                              165170175                                                                     AsnAsnProProGlyArgValLeuThrLeuAlaAlaThrLeuLeuLeu                              180185190                                                                     GlyTrpProLeuTyrLeuAlaPheAsnValSerGlyArgProTyrAsp                              195200205                                                                     ArgPheAlaCysHisTyrAspProTyrGlyProIlePheSerGluArg                              210215220                                                                     GluArgLeuGlnIleTyrIleAlaAspLeuGlyIlePheAlaThrThr                              225230235240                                                                  PheValLeuTyrGlnAlaThrMetAlaLysGlyLeuAlaTrpValMet                              245250255                                                                     ArgIleTyrGlyValProLeuLeuIleValAsnCysPheLeuValMet                              260265270                                                                     IleThrTyrLeuGlnHisThrHisProAlaIleProArgTyrGlySer                              275280285                                                                     SerGluTrpAspTrpLeuArgGlyAlaMetValThrValAspArgAsp                              290295300                                                                     TyrGlyValLeuAsnLysValPheHisAsnIleAlaAspThrHisVal                              305310315320                                                                  AlaHisHisLeuPheAlaThrValProHisTyrHisAlaMetGluAla                              325330335                                                                     ThrLysAlaIleLysProIleMetGlyGluTyrTyrArgTyrAspGly                              340345350                                                                     ThrProPheTyrLysAlaLeuTrpArgGluAlaLysGluCysLeuPhe                              355360365                                                                     ValGluProAspGluGlyAlaProThrGlnGlyValPheTrpTyrArg                              370375380                                                                     AsnLysTyr                                                                     385                                                                           (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 383 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      MetGlyAlaGlyGlyArgMetProValProThrSerSerLysLysSer                              151015                                                                        GluThrAspThrThrLysArgValProCysGluLysProProPheSer                              202530                                                                        ValGlyAspLeuLysLysAlaIleProProHisCysPheLysArgSer                              354045                                                                        IleProArgSerPheSerTyrLeuIleSerAspIleIleIleAlaSer                              505560                                                                        CysPheTyrTyrValAlaThrAsnTyrPheSerLeuLeuProGlnPro                              65707580                                                                      LeuSerTyrLeuAlaTrpProLeuTyrTrpAlaCysGlnGlyCysVal                              859095                                                                        LeuThrGlyIleTrpValIleAlaHisGluCysGlyHisHisAlaPhe                              100105110                                                                     SerAspTyrGlnTrpLeuAspAspThrValGlyLeuIlePheHisSer                              115120125                                                                     PheLeuLeuValProTyrPheSerTrpLysTyrSerHisArgArgHis                              130135140                                                                     HisSerAsnThrGlySerLeuGluArgAspGluValPheValProLys                              145150155160                                                                  GlnLysSerAlaIleLysTrpTyrGlyLysTyrLeuAsnAsnProLeu                              165170175                                                                     GlyArgIleMetMetLeuThrValGlnPheValLeuGlyTrpProLeu                              180185190                                                                     TyrLeuAlaPheAsnValSerGlyArgProTyrAspGlyPheAlaCys                              195200205                                                                     HisPhePheProAsnAlaProIleTyrAsnAspArgGluArgLeuGln                              210215220                                                                     IleTyrLeuSerAspAlaGlyIleLeuAlaValCysPheGlyLeuTyr                              225230235240                                                                  ArgTyrAlaAlaAlaGlnGlyMetAlaSerMetIleCysLeuTyrGly                              245250255                                                                     ValProLeuLeuIleValAsnAlaPheLeuValLeuIleThrTyrLeu                              260265270                                                                     GlnHisThrHisProSerLeuProHisTyrAspSerSerGluTrpAsp                              275280285                                                                     TrpLeuArgGlyAlaLeuAlaThrValAspArgAspTyrGlyIleLeu                              290295300                                                                     AsnLysValPheHisAsnIleThrAspThrHisValAlaHisHisLeu                              305310315320                                                                  PheSerThrMetProHisTyrAsnAlaMetGluAlaThrLysAlaIle                              325330335                                                                     LysProIleLeuGlyAspTyrTyrGlnPheAspGlyThrProTrpTyr                              340345350                                                                     ValAlaMetTyrArgGluAlaLysGluCysIleTyrValGluProAsp                              355360365                                                                     ArgGluGlyAspLysLysGlyValTyrTrpTyrAsnAsnLysLeu                                 370375380                                                                     (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 387 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      MetGlyGlyGlyGlyArgMetSerThrValIleThrSerAsnAsnSer                              151015                                                                        GluLysLysGlyGlySerSerHisLeuLysArgAlaProHisThrLys                              202530                                                                        ProProPheThrLeuGlyAspLeuLysArgAlaIleProProHisCys                              354045                                                                        PheGluArgSerPheValArgSerPheSerTyrValAlaTyrAspVal                              505560                                                                        CysLeuSerPheLeuPheTyrSerIleAlaThrAsnPhePheProTyr                              65707580                                                                      IleSerSerProLeuSerTyrValAlaTrpLeuValTyrTrpLeuPhe                              859095                                                                        GlnGlyCysIleLeuThrGlyLeuTrpValIleGlyHisGluCysGly                              100105110                                                                     HisHisAlaPheSerGluTyrGlnLeuAlaAspAspIleValGlyLeu                              115120125                                                                     IleValHisSerAlaLeuLeuValProTyrPheSerTrpLysTyrSer                              130135140                                                                     HisArgArgHisHisSerAsnIleGlySerLeuGluArgAspGluVal                              145150155160                                                                  PheValProLysSerLysSerLysIleSerTrpTyrSerLysTyrSer                              165170175                                                                     AsnAsnProProGlyArgValLeuThrLeuAlaAlaThrLeuLeuLeu                              180185190                                                                     GlyTrpProLeuTyrLeuAlaPheAsnValSerGlyArgProTyrAsp                              195200205                                                                     ArgPheAlaCysHisTyrAspProTyrGlyProIlePheSerGluArg                              210215220                                                                     GluArgLeuGlnIleTyrIleAlaAspLeuGlyIlePheAlaThrThr                              225230235240                                                                  PheValLeuTyrGlnAlaThrMetAlaLysGlyLeuAlaTrpValMet                              245250255                                                                     ArgIleTyrGlyValProLeuLeuIleValAsnCysPheLeuValMet                              260265270                                                                     IleThrTyrLeuGlnHisThrHisProAlaIleProArgTyrGlySer                              275280285                                                                     SerGluTrpAspTrpLeuArgGlyAlaMetValThrValAspArgAsp                              290295300                                                                     TyrGlyValLeuAsnLysValPheHisAsnIleAlaAspThrHisVal                              305310315320                                                                  AlaHisHisLeuPheAlaThrValProHisTyrHisAlaMetGluAla                              325330335                                                                     ThrLysAlaIleLysProIleMetGlyGluTyrTyrArgTyrAspGly                              340345350                                                                     ThrProPheTyrLysAlaLeuTrpArgGluAlaLysGluCysLeuPhe                              355360365                                                                     ValGluProAspGluGlyAlaProThrGlnGlyValPheTrpTyrArg                              370375380                                                                     AsnLysTyr                                                                     385                                                                           (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1222 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ACAACAGTGAGAAGAAAGGAGGAAGCAGCCACCTTAAGCGAGCGCCGCACACGAAGCCTC60                CTTTCACACTTGGTGACCTCAAGAGAGCCATCCCACCCCATTGCTTTGAACGCTCTTTTG120               TGCGCTCATTCTCCTATGTTGCCTATGATGTCTGCTTAAGTTTTCTTTTCTACTCGATCG180               CCACCAACTTCTTCCCTTACATCTCTTCTCCGCTCTCGTATGTCGCTTGGCTGGTTTACT240               GGCTCTTCCAAGGCTGCATTCTCACTGGTCTTTGGGTCATCGGCCATGAATGTGGCCATC300               ATGCTTTTAGTGAGTATCAGCTGGCTGATGACATTGTTGGCCTAATTGTCCATTCTGCAC360               TTCTGGTTCCATATTTTTCATGGAAATATAGCCATCGCCGCCACCATTCTAACATAGGAT420               CTCTCGAGCGAGACGAAGTGTTCGTCCCGAAATCAAAGTCGAAAATTTCATGGTATTCTA480               AGTACTCAAACAACCCGCCAGGTCGAGTTTTGACACTTGCTGCCACGCTCCTCCTTGGCT540               GGCCTTTATACTTAGCTTTCAATGTCTCTGGTAGACCTTACGATCGCTTTGCTTGCCATT600               ATGATCCCTATGGCCCAATATTTTCCGAAAGAGAAAGGCTTCAGATTTACATTGCTGACC660               TCGGAATCTTTGCCACAACGTTTGTGCTTTATCAGGCTACAATGGCAAAAGGGTTGGCTT720               GGGTAATGCGTATCTATGGGGTGCCATTGCTTATTGTTAACTGTTTCCTTGTTATGATCA780               CATACTTGCAGCACACTCACCCAGCTATTCCACGCTATGGCTCATCGGAATGGGATTGGC840               TCCGGGGAGCAATGGTGACTGTCGATAGAGATTATGGGGTGTTGAATAAAGTATTCCATA900               ACATTGCAGACACTCATGTAGCTCATCATCTCTTTGCTACAGTGCCACATTACCATGCAA960               TGGAGGCCACTAAAGCAATCAAGCCTATAATGGGTGAGTATTACCGGTATGATGGTACCC1020              CATTTTACAAGGCATTGTGGAGGGAGGCAAAGGAGTGCTTGTTCGTCGAGCCAGATGAAG1080              GAGCTCCTACACAAGGCGTTTTCTGGTACCGGAACAAGTATTAAAAAAGTGTCATGTAGC1140              CTGTTTCTTTAAGAGAAGTAATTAGAACAAGAAGGAATGTGTGTGTAGTGTAATGTGTTC1200              TAATAAAGAAGGCAAAAAAAAA1222                                                    (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1231 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CTACTTCTTCCAAGAAATCGGAAACCGACACCACAAAGCGTGTGCCGTGCGAGAAACCGC60                CTTTCTCGGTGGGAGATCTGAAGAAAGCAATCCCGCCGCATTGTTTCAAACGCTCAATCC120               CTCGCTCTTTCTCCTACCTTATCAGTGACATCATTATAGCCTCATGCTTCTACTACGTCG180               CCACCAATTACTTCTCTCTCCTCCCTCAGCCTCTCTCTTACTTGGCTTGGCCACTCTATT240               GGGCCTGTCAAGGCTGTGTCCTAACTGGTATCTGGGTCATAGCCCACGAATGCGGTCACC300               ACGCATTCAGCGACTACCAATGGCTGGATGACACAGTTGGTCTTATCTTCCATTCCTTCC360               TCCTCGTCCCTTACTTCTCCTGGAAGTATAGTCATCGCCGTCACCATTCCAACACTGGAT420               CCCTCGAAAGAGATGAAGTATTTGTCCCAAAGCAGAAATCAGCAATCAAGTGGTACGGGA480               AATACCTCAACAACCCTCTTGGACGCATCATGATGTTAACCGTCCAGTTTGTCCTCGGGT540               GGCCCTTGTACTTAGCCTTTAACGTCTCTGGCAGACCGTATGACGGGTTCGCTTGCCATT600               TCTTCCCCAACGCTCCCATCTACAATGACCGAGAACGCCTCCAGATATACCTCTCTGATG660               CGGGTATTCTAGCCGTCTGTTTTGGTCTTTACCGTTACGCTGCTGCACAAGGGATGGCCT720               CGATGATCTGCCTCTACGGAGTACCGCTTCTGATAGTGAATGCGTTCCTCGTCTTGATCA780               CTTACTTGCAGCACACTCATCCCTCGTTGCCTCACTACGATTCATCAGAGTGGGACTGGC840               TCAGGGGAGCTTTGGCTACCGTAGACAGAGACTACGGAATCTTGAACAAGGTGTTCCACA900               ACATTACAGACACACACGTGGCTCATCACCTGTTCTCGACAATGCCGCATTATAACGCAA960               TGGAAGCTACAAAGGCGATAAAGCCAATTCTGGGAGACTATTACCAGTTCGATGGAACAC1020              CGTGGTATGTAGCGATGTATAGGGAGGCAAAGGAGTGTATCTATGTAGAACCGGACAGGG1080              AAGGTGACAAGAAAGGTGTGTACTGGTACAACAATAAGTTATGAGCATGATGGTGAAGAA1140              ATTGTCGACCTTTCTCTTGTCTGTTTGTCTTTTGTTAAAGAAGCTATGCTTCGTTTTAAT1200              AATCTTATTGTCCATTTTGTTGTGTTATGAC1231                                           (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GCTCTTTTGTGCGCTCATTC20                                                        (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TCGACAGTCACCATTGCTCC20                                                        (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TGGAARTAYWSNCAYMGNMGNCAMCA26                                                  (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AANARRTGRTGNGCNACRTGNGTRTC26                                                  __________________________________________________________________________

What is claimed is:
 1. A seed of a transgenic plant, wherein saidtransgenic plant has integrated in its genome a DNA construct encodingoleate-12 hydroxylase as shown in SEQ ID NO:40, and wherein said DNAconstruct encoding oleate-12 hydroxylase is transcribed and translatedto produce an increased percentage of hydroxylated fatty acids in saidseed as compared to a seed of said plant not containing said DNAconstruct.
 2. The seed of claim 1 wherein said DNA construct containsbase 187 to base 1347 of SEQ ID NO:39.
 3. The seed of claim 1 whereinsaid plant is selected from the group consisting of rapeseed, Canola,flax, sunflower, safflower, cotton, Cuphea, soybean, peanut, coconut,oil palm, and corn.
 4. The seed of claim 1 wherein said DNA constructfurther comprises a seed-specific promoter.
 5. The seed of claim 4wherein said seed-specific promoter is a napin promoter or a promoter ofsaid oleate-12 hydroxylase.
 6. The seed of claim 1 wherein said DNAconstruct further comprises a cauliflbwer mosaic virus 35S promoter. 7.The seed of claim 1 wherein at least one of said hydroxylated fattyacida is selected from the group consisting of 12-hydroxy-9-octadecenoicacid (ricinoleic acid), 14-hydroxy-11-eicosenoic acid (lesquerolicacid), and 12-hydroxy-9,15-octadecadienoic acid.
 8. A seed of atransgenic plant, wherein said transgenic plant has incorporated in itsgenome a DNA construct encoding oleate-12 hydroxylase into said plant,wherein said DNA construct encodes amino acid sequence SEQ ID NO:40, andwherein said DNA construct encoding oleate-12 hydroxylase is transcribedand translated to produce an increased percentage of a hydroxylatedfatty acyl compound in said seed as compared to a seed of said plant notcontaining said DNA construct.
 9. The seed of claim 8 wherein said DNAconstruct contains base 187 to base 1347 of SEQ ID NOL:39.
 10. The seedof claim 8 wherein said plant is selected from the group consisting ofrapeseed, Canola, flax, sunflower, safflower, cotton, Cuphea, soybean,peanut, coconut, oil palm, and corn.
 11. The seed of claim 8 whereinsaid DNA construct further comprises a seed-specific promoter.
 12. Theseed of claim 11 wherein said seed-specific promoter is a napin promoteror a promoter of said oleate-12 hydroxylase.
 13. The seed of claim 8wherein said DNA construct further comprises a cauliflower mosaic virus35S promoter.
 14. The seed of claim 8 wherein said hydroxylated fattyacyl compound comprises a hydroxylated fatty acid selected from thegroup consisting of 12-hydroxy-9-octadecenoic acid (ricinoleic acid),14-hydroxy-11-eicosenoic acid (lesquerolic acid), and12-hydroxy-9,15-octadecadienoic acid.